Microsatellite Markers of Schizophrenia

ABSTRACT

The invention includes methods of determining if a subject is at risk for developing schizophrenia (SZ).

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No.11/323,061, filed on Dec. 30, 2005, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/640,707, filed on Dec. 30,2004. The entire contents of the foregoing are hereby incorporated byreference.

STATE SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with an award from the Kentucky Scienceand Technology Corporation under Contract No. 144-401-06.

TECHNICAL FIELD

This invention relates to genetic markers of schizophrenia, and methodsof use thereof.

BACKGROUND

Numerous linkage and association studies have implicated chromosome 22qin the etiology of schizophrenia (Vallada et al., Psychiatr. Genet.5:127-30 (1995); Gill et al., Am. J. Med. Genet. 16:40-5 (1996);Myles-Worsley et al., Am. J. Med. Genet. 88:544-50 (1999); Jorgensen etal., Am. J. Med. Genet. 114:245-52 (2002); DeLisi et al., Am. J.Psychiatry 159:803-12 (2002); Lewis et al., Am. J. Hum. Genet. 73:34-48(2003); Takahashi et al., Am. J. Med. Genet. 120B:11-7 (2003)).Nonetheless, the precise location of the genes involved has yet to beresolved.

Possibly owing to genetic heterogeneity, analyses of positionalcandidates on this chromosome have resulted in conflicting results. The22q11 region has received much attention, as its deletion invelo-cardio-facial syndrome correlates with increased propensity todevelop schizophrenia (Ivanov et al., Br J Psychiatry. 183:409-13(2003); van Amelsvoort et al., Genetic Curr. Psychiatry. Rep. 6:176-82(2004); Williams and Owen, Curr Psychiatry Rep. 6(3):176-82 (2004)).Candidates identified in this region include thecatechol-O-methyltransferase (COMT) gene, an attractive candidate whoserole has recently been challenged, and proline dehydrogenase, a genewhose role may be limited to Chinese lineages (Shifman et al., Am. J.Hum. Genet. 71:1296-302 (2002); Williams and Owen, (2004), supra;McGuffin et al., Curr. Psychiatry. Rep. 5:121-7 (2003); Williams et al.,Am. J. Med. Genet. 120B:42-6 (2003); Handoko et al., Mol. Psychiatry.10:589-597 (2005) [Epub ahead of print Oct. 26, 2004]; Shirts andNimgaonkar, Curr. Psychiatry. Rep. 6:303-12 (2004)). Other studiessuggest a more distal location for a susceptibility gene in 22q12 or22q13 (DeLisi et al., 2002, supra; Takahashi et al., Am. J. Med. Genet.120B:11-7 (2003) et al., 2003). Here again, however, family-basedtransmission studies and evaluation of specific candidate genes haveprovided somewhat modest or, at times, contradictory, results (Valladaet al., Psychiatr. Genet. 5:127-30 (1995); Stober et al., Am. J. Med.Genet. 96:392-7 (2000); Meyer et al., Mol. Psychiatry. 6:302-6 (2001);Takahashi et al., Am. J. Med. Genet. 120B:11-7 (2003); Georgieva et al.,Psychiatr. Genet. 13:103-6 (2003); Kaganovich et al., Am. J. Med. Genet.125B:31-7 (2004)).

Due to the severity of the disorder, the negative impact of a psychoticepisode on a patient, and the diminishing recovery after each psychoticepisode, there is a need to more conclusively identify individuals whohave or are at risk of developing schizophrenia (SZ), schizotypalpersonality disorder (SPD) or schizoaffective disorder (SD), forexample, to confirm clinical diagnoses, to allow for prophylactictherapies, to determine appropriate therapies based on their genotypicsubtype, and to provide genetic counseling for prospective parents witha history of the disorder.

SUMMARY

In previous work the present inventors developed a high quality linkagegenetic map of chromosome 22 that included two extreme distal markers(Brennan et al., Genomics 63:430-432 (2000); Matise et al., Am. J. Hum.Genet. 70:1398-410 (2002)). These and other highly informativemicrosatellite markers (including a new microsatellite marker targetingthe promoter region of the Sult4a1 gene) were used to evaluated 27families from the NIMH Schizophrenia Genetics Initiative. Based on thelinkage and family-based association patterns that were observed, amulti-locus model involving at least the Sult4A1 region and a moredistal region near marker D22s256 in 22q13 is described herein. Thus,the invention includes methods of determining risk of developingschizophrenia (SZ), schizotypal personality disorder (SPD) orschizoaffective disorder (SD) as described herein.

In one aspect, the invention includes methods for obtaining informationregarding a subject's risk for developing SZ, SD or SPD. The methodsinclude obtaining a test haplotype associated with schizophrenia asdescribed herein. The methods can also include obtaining a samplecomprising genomic DNA (gDNA) from the subject, and determining theidentity, absence or presence of a test haplotype associated with SZ, SDor SPD as described herein. In some embodiments, the methods includeobtaining a test haplotype for the subject comprising at least one testmarker that is within 1 linkage disequilibrium unit (1 LDU) of a markerlisted in Table 4, 6, 7, 8 or 9, wherein the haplotype providesinformation regarding the subject's risk of developing SZ, SPD, or SD.In some embodiments, the test marker is a marker listed in one or moreof tables 4, 6, 7, 8, or 9, or a marker within 1 linkage disequilibriumunit (1 LDU) or >0.5 D′ of a polymorphism described herein, e.g.,markers in a region of chromosome 22, e.g., in 22q13, e.g., in 22q13.3,that is between and including SNPs rs738596, rs738598, or rs135221 onthe proximal end, and rs13884 or rs137853 on the distal end, e.g.,between rs738596 and rs137853.

In some embodiments, the test marker is within 1 LDU of a marker listedin Table 6, 7, 8, or 9, and is in a region of 22q13 that is between andincluding SNPs rs738596, rs738598, or rs135221 on the proximal end, andrs137853 or rs13884 on the distal end.

In some embodiments, the test haplotype includes at least one markerlisted in Table 4, 6, 7, 8 or 9.

In some embodiments, the test haplotype includes one or more of:microsatellite marker D22S526, and/or a polymorphism of Sulfotransferase4A1 (Sult4a1), e.g., rs138060, rs138097, rs138110, and/or D22s1749e. Insome embodiments, the polymorphism is an allele of Sult4a1 atmicrosatellite marker D22s1749e comprising more than 207 nucleotides,and indicates that the subject has an increased risk of developing SZ,SPD, or SD.

In some embodiments, the test haplotype includes at least two markers,one of which is microsatellite marker D22S526.

In some embodiments, the test haplotype includes at least one markerlisted in Table 4 or 9, or in bold in table 8, and provides informationregarding a subject's risk of developing SZ, under a narrower (DSM III)disease definition.

The methods described herein can include obtaining a haplotype thatincludes two or more, e.g., two, three, four, five, or six markers.

Additionally, the methods can include determining the presence orabsence of other markers known to be associated with SZ, SD or SPD,e.g., outside of a region identified herein. A number of other suchmarkers are known in the art, e.g., as described herein.

The subject can be a mammal, e.g., a primate, preferably a higherprimate, e.g., a human (e.g., a patient having, or at risk of, SZ, SD orSPD). In one embodiment, the subject is a patient having SZ, SD or SPD(e.g., a patient suffering from early, intermediate or aggressive SZ, SDor SPD). In some embodiments, the methods described herein are used toobtain information regarding a subject's risk of developing SZ, SD orSPD, wherein the disorder is other than catatonic schizophrenia. In someembodiments, the subject is of African American (AA) or EuropeanAmerican (EA) descent, i.e., has one or more ancestors who are AA or EA.

In one embodiment, a subject to be evaluated by a method describedherein is a subject having one or more risk factors associated with SZ,SPD or SD. For example, the subject may have a relative afflicted withSZ, e.g., one or more of a grandparent, parent, uncle or aunt, sibling,or child who has or had SZ, SPD or SD; the subject may have agenetically based phenotypic trait associated with risk for SZ, SPD orSD (e.g., eye tracking dysfunction); deficits in working (short-term)memory; and/or mixed-handedness (the use of different hands fordifferent tasks), particularly in females.

In some embodiments, the subject is a child, fetus, or embryo, and oneof the subject's relatives, e.g., a parent or sibling, of the child,fetus, or embryo has SZ, SPD or SD. In this case, the presence in thechild, fetus, or embryo of a haplotype described herein that is sharedwith the affected parent, but not with the non-affected parent,indicates that the child, fetus, or embryo has an increased risk ofdeveloping SPD, SD, or SZ. In some embodiments, the subject has no overtor clinical signs of SZ, SPD, or SD.

In some embodiments, obtaining a test haplotype includes obtaining asample comprising DNA from the subject; and determining the identity,presence or absence of at least one test marker that is within 1 LDU ofa marker listed in Table 4, 6, 7, 8 or 9 in the DNA. The sample can beobtained, e.g., from the subject by a health care provider, or providedby the subject without the assistance of a health care provider.

In some embodiments, obtaining a test haplotype includes reviewing asubject's medical history, wherein the medical history includesinformation regarding the presence or absence of at least one testmarker that is within 1 LDU of a marker listed in Table 4, 6, 7, 8 or 9in the subject.

In some embodiments, the methods described herein include obtaining areference haplotype including a reference marker that corresponds to atest marker, and comparing the test haplotype to the referencehaplotype. A reference marker that “corresponds to” a test marker is thesame marker. For example, if the test haplotype includes D22S526, thenthe reference haplotype should also include D22S526 for comparisonpurposes. The sharing of a haplotype (e.g., of some or all of themarkers) between the test haplotype and a reference haplotype isindicative of whether there is an increased likelihood that the subjectwill develop SZ, SPD, or SD.

In some embodiments, the methods include administering a treatment to asubject identified as being at increased risk for developing SZ, SPD, orSD, e.g., a pharmacological or psychosocial treatment as describedherein. In some embodiments, the subject has no overt or clinical signsof SZ, SPD, or SD, and the treatment is administrated before any suchsigns appear.

Information obtained using a method described herein can be used, e.g.,to select a subject population for a clinical trial, to stratify asubject population in a clinical trial, and/or to stratify subjects thatrespond to a treatment from those who do not respond to a treatment, orsubjects that have negative side effects from those who do not.

In another aspect, the invention provides methods for selecting asubject for inclusion in a clinical trial, e.g., a trial of a treatmentfor SZ, SPD, or SD. The methods include obtaining a haplotype for thesubject including at least one marker that is within 1 linkagedisequilibrium unit (1 LDU) of a marker listed in Tables 4, 6, 7, 8 or9; determining whether the haplotype is associated with an increasedrisk of developing schizophrenia (SZ), schizotypal personality disorder(SPD), or schizoaffective disorder (SD); and including the subject inthe trial if the haplotype indicates that the subject has an increasedrisk of developing SZ, SPD, or SD.

In another aspect, the invention provides methods for selecting asubject for administration of a treatment for schizophrenia (SZ),schizotypal personality disorder (SPD), or schizoaffective disorder(SD). The methods include obtaining a haplotype for the subject, whereinthe haplotype comprises at least one marker that is within 1 linkagedisequilibrium unit (1 LDU) of a marker listed in Tables 4, 6, 7, 8 or9; determining whether the haplotype is associated with an increasedrisk of developing SZ, SPD, or SD; and administering the treatment tothe subject if the haplotype indicates that the subject has an increasedrisk of developing SZ, SPD, or SD.

In another aspect, the invention provides methods for selecting atreatment for administration to a subject. The methods include obtaininga haplotype for the subject, wherein the haplotype comprises at leastone marker that is within 1 linkage disequilibrium unit (1 LDU) of amarker listed in Tables 4, 6, 7, 8 or 9; determining whether thehaplotype is associated with an increased risk of developingschizophrenia (SZ), schizotypal personality disorder (SPD), orschizoaffective disorder (SD); and administering the treatment for SZ,SPD, or SD to the subject if the haplotype indicates that the subjecthas an increased risk of developing SZ, SPD, or SD.

In another aspect, the invention provides methods for evaluating theeffect of a haplotype on the outcome of a treatment for schizophrenia(SZ), schizotypal personality disorder (SPD), or schizoaffectivedisorder (SD). The methods include obtaining information regardingoutcome of the treatment, wherein the information comprises a parameterrelating to the treatment of each subject in a population of subjects;obtaining haplotypes for each subject in the population, wherein thehaplotype comprises at least one marker that is within 1 linkagedisequilibrium unit (1 LDU) of a marker listed in Tables 4, 6, 7, 8 or9; and correlating the information regarding outcome with thehaplotypes; thereby evaluating the effect of the haplotype on theoutcome of the treatment.

In some embodiments, the method includes selecting a treatment foradministration to a subject who has a selected haplotype, based on theeffect of the haplotype on the outcome of the treatment.

In some embodiments, the information regarding outcome of the treatmentis from a completed clinical trial, and the analysis is retrospective.

In another aspect, the invention features methods of predicting asubject's risk of developing SZ, SPD, or SD. The methods includeobtaining a reference haplotype. In some embodiments, the referencehaplotype is from at least one of the following relatives of thesubject: (i) a parent who has SZ, SPD, or SD; (ii) a sibling who has SZ,SPD, or SD, and an unaffected parent; or (iii) a second degree relative(e.g., aunt, uncle, or grandparent) who has SZ, SPD, or SD, and anunaffected parent; obtaining a test haplotype from the subject in thesame region; and comparing the test haplotype to a reference haplotype.The sharing of a haplotype in this region between the test haplotype anda reference haplotype from a relative having the disorder is anindication of an increased likelihood that the subject will develop SZ,SPD, or SD. In some embodiments, the reference haplotype is from anunaffected individual, and sharing of a haplotype indicates that thereis no increased likelihood that the subject will develop SZ, SD, or SD.

In a further aspect, the invention features methods for detecting thepresence of a haplotype associated with susceptibility to SZ, SPD, or SDin a subject, by analyzing a sample of DNA from the subject.

Additionally, the invention features methods of predicting a testsubject's risk of developing SZ, SPD, or SD. The methods includeobtaining a reference haplotype of a reference subject, wherein thereference subject has SZ, SPD, or SD; determining a test haplotype ofthe test subject in the same region; and comparing the test haplotype tothe reference haplotype, wherein the sharing of a haplotype in thisregion between the test subject and the reference subject is anindication of an increased likelihood that the test subject will developSZ, SPD, or SD. In some embodiments, the method further includescomparing the subject's haplotype to a reference subject who does nothave SZ, SPD, or SD.

Further, the invention features methods for predicting a test subject'srisk of developing SZ. The methods include obtaining a referencehaplotype of a reference subject in a region described herein, whereinthe reference subject has SZ; obtaining a test haplotype of the testsubject in the same region; and comparing the test haplotype to thereference haplotype. The sharing of a haplotype in this region betweenthe test subject and the reference subject is an indication of anincreased likelihood that the test subject will develop SZ. In someembodiments, the method also includes comparing the test subject'shaplotype to a reference subject who does not have SZ.

In another aspect, the invention features methods for predicting asubject's risk of developing SZ, SPD, or SD. The methods includeobtaining genomic DNA (gDNA) from the subject; and determining theabsence or presence of a haplotype associated with SZ at humanchromosome 22q13 as described herein. The presence of a haplotypeassociated with SZ, SPD, or SD indicates that the subject has anincreased risk of developing SZ, SD or SPD.

The invention further features nucleic acid probes having a nucleotidesequence that hybridizes with a nucleotide sequence within humanchromosome 22q13 and allows detection of a microsatellite marker atD22s1749E, e.g., under hybridization conditions of a 50% formamide,2×SSC wash for 10 minutes at 45° C. followed by a 2×SSC wash for 10minutes at 37° C. In some embodiments, the probes are at least 20nucleotides long and include all or part of5′-CAGCCGCACGCCATGGAACTCGAAG-3′(SEQ ID NO:1) or5′-GGCGCCATGACGTCACGCCTGC-3′ (SEQ ID NO:2). In some embodiments, theprobes are no longer than 30, 50, 100, 200, or 500 nucleotides long.

Also provided herein are kits for use in detection of haplotypesassociated with SZ, SD or SPD, including at least one nucleic acid probethat hybridizes to a sequence that includes a polymorphism describedherein, or can be used to amplify a sequence that includes apolymorphism described herein.

Also provided are arrays that include a substrate having a plurality ofaddressable areas, wherein one or more of the addressable areas includesone or more probes that can be used to detect a polymorphism describedherein.

In another aspect, the invention provides methods for providinginformation regarding a subject's risk of developing schizophrenia (SZ),schizotypal personality disorder (SPD), or schizoaffective disorder(SD). The methods include obtaining a sample from the subject at a firstsite; transferring the sample to a second site for analysis, wherein theanalysis provides data regarding the identity, presence or absence of atleast one test marker that is within 1 LDU of a marker listed in Tables4, 6, 7, 8 or 9; and transferring the data to one or more of a healthcare provider, the subject, or a healthcare payer. In some embodiments,the first site is a health care provider's place of business, or is nota health care provider's place of business, e.g., the subject's home.

In some embodiments, the data is transferred to a healthcare payer andused to decide whether to reimburse a health care provider.

DEFINITIONS

As used herein, a “haplotype” is a set of signature genetic changes(polymorphisms) that are normally grouped closely together on the DNAstrand, and are usually inherited as a group; the polymorphisms are alsoreferred to herein as “markers.” A “haplotype” as used herein isinformation regarding the presence or absence of one or more geneticmarkers in a subject. A haplotype can consist of a variety of geneticmarkers, including indels (insertions or deletions of the DNA atparticular locations on the chromosome); single nucleotide polymorphisms(SNPs) in which a particular nucleotide is changed; microsatellites; andminisatellites.

Microsatellites (sometimes referred to as a variable number of tandemrepeats or VNTRs) are short segments of DNA that have a repeatedsequence, usually about 2 to 5 nucleotides long (e.g., CACACA), thattend to occur in non-coding DNA. Changes in the microsatellitessometimes occur during the genetic recombination of sexual reproduction,increasing or decreasing the number of repeats found at an allele,changing the length of the allele. Microsatellite markers are stable,polymorphic, easily analyzed and occur regularly throughout the genome,making them especially suitable for genetic analysis.

“Linkage disequilibrium” refers to when the observed frequencies ofhaplotypes in a population does not agree with haplotype frequenciespredicted by multiplying together the frequency of individual geneticmarkers in each haplotype.

The term “chromosome” as used herein refers to a gene carrier of a cellthat is derived from chromatin and comprises DNA and protein components(e.g., histones). The conventional internationally recognized individualhuman genome chromosome numbering identification system is employedherein. The size of an individual chromosome can vary from one type toanother with a given multi-chromosomal genome and from one genome toanother. In the case of the human genome, the entire DNA mass of a givenchromosome is usually greater than about 100,000,000 base pairs. Forexample, the size of the entire human genome is about 3×10⁹ base pairs.Chromosome 22 contains about 5.3×10⁷ base pairs (see, e.g., Yunis,Science 191:1268-1270 (1976), and Kavenoff et al., Cold Spring HarborSymposia on Quantitative Biology 38:1-8 (1973)).

The term “gene” refers to a DNA sequence in a chromosome that codes fora product (either RNA or its translation product, a polypeptide). A genecontains a coding region and includes regions preceding and followingthe coding region (termed respectively “leader” and “trailer”). Thecoding region is comprised of a plurality of coding segments (“exons”)and intervening sequences (“introns”) between individual codingsegments.

The term “probe” refers to an oligonucleotide. A probe can be singlestranded at the time of hybridization to a target. As used herein,probes include primers, i.e., oligonucleotides that can be used to primea reaction, e.g., a PCR reaction.

The term “label” or “label containing moiety” refers in a moiety capableof detection, such as a radioactive isotope or group containing same,and nonisotopic labels, such as enzymes, biotin, avidin, streptavidin,digoxygenin, luminescent agents, dyes, haptens, and the like.Luminescent agents, depending upon the source of exciting energy, can beclassified as radioluminescent, chemiluminescent, bioluminescent, andphotoluminescent (including fluorescent and phosphorescent). A probedescribed herein can be bound, e.g., chemically bound tolabel-containing moieties or can be suitable to be so bound. The probecan be directly or indirectly labeled.

The term “direct label probe” (or “directly labeled probe”) refers to anucleic acid probe whose label after hybrid formation with a target isdetectable without further reactive processing of hybrid. The term“indirect label probe” (or “indirectly labeled probe”) refers to anucleic acid probe whose label after hybrid formation with a target isfurther reacted in subsequent processing with one or more reagents toassociate therewith one or more moieties that finally result in adetectable entity.

The terms “target,” “DNA target,” or “DNA target region” refers to anucleotide sequence that occurs at a specific chromosomal location. Eachsuch sequence or portion is preferably at least partially, singlestranded (e.g., denatured) at the time of hybridization. When the targetnucleotide sequences are located only in a single region or fraction ofa given chromosome, the term “target region” is sometimes used. Targetsfor hybridization can be derived from specimens which include, but arenot limited to, chromosomes or regions of chromosomes in normal,diseased or malignant human cells, either interphase or at any state ofmeiosis or mitosis, and either extracted or derived from living orpostmortem tissues, organs or fluids; germinal cells including sperm andegg cells, or cells from zygotes, fetuses, or embryos, or chorionic oramniotic cells, or cells from any other germinating body; cells grown invitro, from either long-term or short-term culture, and either normal,immortalized or transformed; inter- or intraspecific hybrids ofdifferent types of cells or differentiation states of these cells;individual chromosomes or portions of chromosomes, or translocated,deleted or other damaged chromosomes, isolated by any of a number ofmeans known to those with skill in the art, including libraries of suchchromosomes cloned and propagated in prokaryotic or other cloningvectors, or amplified in vitro by means well known to those with skill;or any forensic material, including but not limited to blood, or othersamples.

The term “hybrid” refers to the product of a hybridization procedurebetween a probe and a target.

The term “hybridizing conditions” has general reference to thecombinations of conditions that are employable in a given hybridizationprocedure to produce hybrids, such conditions typically involvingcontrolled temperature, liquid phase, and contact between a probe (orprobe composition) and a target. Conveniently and preferably, at leastone denaturation step precedes a step wherein a probe or probecomposition is contacted with a target. Guidance for performinghybridization reactions can be found in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (2003),6.3.1-6.3.6. Aqueous and nonaqueous methods are described in thatreference and either can be used. Hybridization conditions referred toherein are a 50% formamide, 2×SSC wash for 10 minutes at 45° C. followedby a 2×SSC wash for 10 minutes at 37° C.

Calculations of “identity” between two sequences can be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondnucleic acid sequence for optimal alignment and non-identical sequencescan be disregarded for comparison purposes). The length of a sequencealigned for comparison purposes is at least 30%, e.g., at least 40%,50%, 60%, 70%, 80%, 90% or 100%, of the length of the referencesequence. The nucleotides at corresponding nucleotide positions are thencompared. When a position in the first sequence is occupied by the samenucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position. The percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences, taking into account the number ofgaps, and the length of each gap, which need to be introduced foroptimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In some embodiments, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package, using a Blossum 62 scoring matrix with a gap penaltyof 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

As used herein, the term “substantially identical” is used to refer to afirst nucleotide sequence that contains a sufficient number of identicalnucleotides to a second nucleotide sequence such that the first andsecond nucleotide sequences have similar activities. Nucleotidesequences that are substantially identical are at least 80%, e.g., 85%,90%, 95%, 97% or more, identical.

The term “nonspecific binding DNA” refers to DNA which is complementaryto DNA segments of a probe, which DNA occurs in at least one otherposition in a genome, outside of a selected chromosomal target regionwithin that genome. An example of nonspecific binding DNA comprises aclass of DNA repeated segments whose members commonly occur in more thanone chromosome or chromosome region. Such common repetitive segmentstend to hybridize to a greater extent than other DNA segments that arepresent in probe composition.

As used herein, the term “stratification” refers to the creation of adistinction between subjects on the basis of a characteristic orcharacteristics of the subjects. Generally, in the context of clinicaltrials, the distinction is used to distinguish responses or effects indifferent sets of patients distinguished according to the stratificationparameters. In some embodiments, stratification includes distinction ofsubject groups based on the presence or absence of particular markers orhaplotypes described herein. The stratification can be performed, e.g.,in the course of analysis, or can be used in creation of distinct groupsor in other ways.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a line graph illustrating LOD scores for markers at theindicated locations on the long arm of chromosome 22. The locations ofmarkers D22s683, D22s270, and sJCW16, which are associated with thehighest LOD scores, are shown.

FIG. 2 is a line graph illustrating LOD scores for markers at theindicated locations on the long arm of chromosome 22, including the newmarker D22S1749E, the location of which is indicated.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The methods described herein are based, at least in part, on thediscovery of haplotypes and markers in 22q13 that are associated withincreased risk of developing schizophrenia (SZ), schizotypal personalitydisorder (SPD) or schizoaffective disorder (SD). As described herein,TDT analysis provided suggestive evidence of role of Sult4a1 in this setof families (P=0.002 for narrowly-defined SZ, 0.04 for SZ+SPD), with atendency for one of the longer alleles of D22S1749E to be preferentiallytransferred to affected children (See Examples, below). Additionally,TDT analysis for the other microsatellite markers suggests that a regionnear marker D22S526 plays a role in SZ (P=0.003, for narrowly-definedSZ; P=0.00009 for SZ+SD+SPD). Thus, segments of chromosome 22 nearSult4a1 and D22S526 contain sequences that are linked to apredisposition to SPD, SD and SZ.

Methods of Diagnoses and Evaluation of Risk

Described herein are a variety of methods for the diagnosis ofsusceptibility to SZ, SPD or SD. “Susceptibility” does not necessarilymean that the subject will develop SZ, SPD or SD, but rather that thesubject is, in a statistical sense, more likely to develop SZ than anaverage member of the population, i.e., has an increased risk ofdeveloping SZ, SPD, or SD. As used herein, susceptibility to SZ existsif the subject has a haplotype associated with an increased risk of SZ,SPD, or SD as described herein. Ascertaining whether the subject hassuch a haplotype is included in the concept of diagnosing susceptibilityto SZ, SPD or SD as used herein. Such determination is useful, forexample, for purposes of diagnosis, treatment selection, and geneticcounseling. Thus, the methods described herein can include obtaining ahaplotype associated with an increased risk of SZ, SPD, or SD asdescribed herein for the subject.

As used herein, “obtaining a haplotype” includes obtaining informationregarding the identity, presence or absence of one or more geneticmarkers in a subject. Obtaining a haplotype can, but need not, includeobtaining a sample comprising DNA from a subject, and/or assessing theidentity, presence or absence of one or more genetic markers in thesample. The individual or organization who obtains the haplotype neednot actually carry out the physical analysis of a sample from a subject;the haplotype can include information obtained by analysis of the sampleby a third party. Thus the methods can include steps that occur at morethan one site. For example, a sample can be obtained from a subject at afirst site, such as at a health care provider, or at the subject's homein the case of a self-testing kit. The sample can be analyzed at thesame or a second site, e.g., at a laboratory or other testing facility.

Obtaining a haplotype can also include or consist of reviewing asubject's medical history, where the medical history includesinformation regarding the identity, presence or absence of one or moregenetic markers in the subject, e.g., results of a genetic test.

In some embodiments, to detect the presence of a haplotype describedherein, a biological sample that includes nucleated cells (such asblood, a cheek swab or mouthwash) is prepared and analyzed for thepresence or absence of preselected markers. Such diagnoses may beperformed by diagnostic laboratories, or, alternatively, diagnostic kitscan be manufactured and sold to health care providers or to privateindividuals for self-diagnosis. Diagnostic or prognostic tests can beperformed as described herein or using well known techniques, such asdescribed in U.S. Pat. No. 5,800,998.

Results of these tests, and optionally interpretive information, can bereturned to the subject, the health care provider or to a third partypayor. The results can be used in a number of ways. The information canbe, e.g., communicated to the tested subject, e.g., with a prognosis andoptionally interpretive materials that help the subject understand thetest results and prognosis. The information can be used, e.g., by ahealth care provider, to determine whether to administer a specificdrug, or whether a subject should be assigned to a specific category,e.g., a category associated with a specific disease endophenotype, orwith drug response or non-response. The information can be used, e.g.,by a third party payor such as a healthcare payer (e.g., insurancecompany or HMO) or other agency, to determine whether or not toreimburse a health care provider for services to the subject, or whetherto approve the provision of services to the subject. For example, thehealthcare payer may decide to reimburse a health care provider fortreatments for SZ, SPD or SD if the subject has an increased risk ofdeveloping SZ, SPD or SD. As another example, a drug or treatment may beindicated for individuals with a certain haplotype, and the insurancecompany would only reimburse the health care provider (or the insuredindividual) for prescription or purchase of the drug if the insuredindividual has that haplotype. The presence or absence of the haplotypein a patient may be ascertained by using any of the methods describedherein.

Information gleaned from the methods described herein can also be usedto select or stratify subjects for a clinical trial. For example, thepresence of a selected haplotype described herein can be used to selecta subject for a trial. The information can optionally be correlated withclinical information about the subject, e.g., diagnostic orendophenotypic information.

Haplotypes Associated with SZ, SPD and SD

As described herein, haplotypes associated with SZ, SPD or SD includemarkers in the distal region of the long arm of chromosome 22 (i.e., in22q13.3) as exemplified by the transmission disequilibrium results shownin tables 4, 6, 7, 8 or 9.

As one example, haplotypes associated with a broader disorder definitionincluding SZ, SPD and SD include one or more markers on chromosome 22that are within 1 linkage disequilibrium unit (1 LDU) of a marker listedin Tables 4, 6, 7, 8 or 9. In some embodiments, the haplotype includesone or more of the markers listed in tables 4, 6, 7, 8 or 9. In someembodiments, the markers are in a region of 22q13 that is between andincludes SNPs rs738596, rs738598, or rs135221 on the proximal end, andrs13884 or rs137853 on the distal end. In some embodiments, the markersare in a region of 22q13 that is between and includes SNPs rs738598 andrs13884. In some embodiments, the markers are in a region of 22q13 thatis between and includes SNPs rs135221 and rs13884.

Haplotypes associated with a narrower disorder definition of SZ caninclude one or more markers that are within 1 LDU of a marker listed inTable 4 or 9, or in bold in table 8. In some embodiments, the haplotypeincludes one or more of the markers listed in Tables 4 or 9, or in boldin table 8. In some embodiments, the markers are in a region of 22q13that is between and includes rs135221 on the proximal end, and rs13884on the distal end.

In some embodiments, the methods include determining the presence of ahaplotype that includes one or more polymorphisms near D22S526 and/orthe polymorphisms in the Sult4a1 gene listed in Table 4, and/orpolymorphisms within 1 LDU of these markers.

In some embodiments, the methods described herein do not includedetecting polymorphisms within the MLC1 gene.

Sulfotransferase-4A1 (Sult4a1)

Using samples obtained from the National Institutes of Mental HealthSchizophrenia Genetics Initiative, 27 nuclear families having multiplesiblings with schizophrenia and schizophrenia-spectrum disorders wereevaluated for linkage to chromosome 22 markers. Analysis with 14 highlyinformative microsatellite markers provided evidence for linkage nearmarker D22s270. Assuming heterogeneity, a maximum LOD score of 2.90 wasobtained using DSM IV criteria, and a maximum LOD score of 3.96 wasobtained for a broader disease definition that included schizotypalpersonality disorder (SPD). Nonparametric linkage analysis providedsuggestive evidence for linkage at the same location (LOD scores of 2.6and 2.8 for the narrow and broad definitions, respectively).

This segment of chromosome 22 contains the sulfotransferase-4A1(Sult4a1) gene, which encodes a brain-specific sulfotransferase believedto be involved in metabolism of neurotransmitters (Falany et al.,Biochem J. 346:857-64 (2000); Sakakibara et al., Gene 285:39-47 (2002);Liyou et al., J. Histochem. Cytochem. 51:655-64 (2003)). This positionalcandidate was evaluated by family-based TDT analysis of 27 families fromthe NIMH Schizophrenia Genetics Initiative. To evaluate this candidategene, a microsatellite marker (D22S1749E) targeting a promoterpolymorphism in the gene was developed, and transmission disequilibrium(TDT) analysis of this marker and three single nucleotide polymorphismsspanning a 37 kb region containing the gene was performed.

As described herein, TDT analysis provided suggestive evidence of roleof Sult4a1 in this set of families (P=0.002 for narrowly-defined SZ,0.04 for SZ+SPD), with a tendency for one of the longer alleles (213 nt)of D22S1749E to be preferentially transferred to affected children (SeeExamples 1-4, below).

The sample was expanded by the addition of 17 further families to theoriginal 27 families. Using the D22S1749E marker in linkage analysis forthe pooled sample (using a dominant model assuming geneticheterogeneity, a penetrance of 50% for a heterozyote and a 1% allelefrequency) a single point heterogeneous LOD score of 4.78 was obtainedfor the combined sample of 44 families (α=0.7). Consistent with theinitial findings, for the pooled sample, D22S1749E shows significantdeviation from expectation for transmission to affected offspring usingTRANSMIT (P=0.015 for SZ, and P=0.006 for the broader definitionincluding SPD).

Thus, the methods described herein can include detecting the identity,presence or absence of one or more polymorphisms of the Sult4a1 gene,e.g., polymorphisms described herein. For example, the methods describedherein can include determining the presence of a polymorphism atD22S1749E, e.g., determining the length of the alleles at D22S1749E. Insome embodiments the methods also include detecting the presence of aSNP in the Sult4a1 gene, e.g., one or more of rs138060, rs138097, andrs138110 (see, e.g., Example 3 and Table 4).

D22S526 and the Distal Region of 2203

Numerous two and three SNP haplotypes spanning the distal region of22q13 show highly significant distortions in transmission ratios forDSM-IIIR diagnosed SZ and broader disease definitions (see the Examples,below; P<10⁻⁵). Some of these remain significant even after the mostparsimonious corrections for multiple comparisons (Risch and Merikangas,Science 273(5281):1516-7 (1996); Sabatti et al., Genetics 164(2):829-33(2003)). One SNP by itself, rs1573726, shows significant TDT values bythis method (X²=15.6, 1df, P=7.8×10⁻⁵).

Thus, the methods described herein include identifying subjects on thebasis of having a haplotype that includes polymorphisms that are in theregion of chromosome 22 that is defined by the SNPs rs738596 (on theproximal end) and rs137853 (on the distal end). In some embodiments, themethods include identifying haplotypes that include polymorphismsbetween SNPs rs738598 (proximal) and rs137853 or rs138844 (distal), orbetween rs135221 (proximal) and rs137853 or rs138844 (distal). Proximalrefers to a location that is nearer the centromere, distal is furtheraway. In some embodiments, the methods do not include the evaluation ofpolymorphisms at microsatellite D22s1169.

A close evaluation of the haplotypes revealed an interesting pattern.Indeed, there are particular SNP haplotypes preferentially transmitted,and these differ somewhat in EA (European American) and AA (AfricanAmerican) families. Some are not rare, but fairly common haplotypeshaving 25 to 40% expected frequencies based on information now availablethrough the haplotyping consortium (on the world wide web athapmap.org). However, in about half of the NIMH families, these SNPhaplotypes occur as part of a larger haplotype involving a small subset(two to four per population) of the 23 alleles of the highly polymorphicmarker D22s526. TDT analysis for the other microsatellite markerssuggests that a region near marker D22S526 plays a role in SZ (P=0.003,for narrowly-defined SZ; P=0.00009 for SZ+SPD+SD), possibly due tomicrodeletions of the region immediately surrounding and including thishighly polymorphic marker (see Examples 4 and 7, below). Thus, in someembodiments, the methods described herein include the evaluation ofpolymorphisms of D22S526, to detect microdeletions e.g., microdeletionsthat include D22S526, e.g., microdeletions of at least 50, 100, 200,300, 400, 500 or more Kb. In some embodiments, the microdeletions appearas apparent homozygosity, and the presence of homozygosity at D22S526 isindicative of an increased risk of developing SZ, SD, or SPD.

Linkage Disequilibrium Analysis

Linkage disequilibrium (LD) is a measure of the degree of associationbetween alleles in a population. One of skill in the art will appreciatethat haplotypes involving markers within 1 Linkage Disequilibrium Unit(LDU) of the polymorphisms described herein can also be used in asimilar manner to those described herein. LDUs share an inverserelationship with LD so that regions with high LD (such as haplotypeblocks) have few LDUs and low recombination, whilst regions with manyLDUs have low LD and high recombination. Methods of calculating LDUs areknown in the art (see, e.g., Morton et al., Proc Natl Acad Sci USA98(9):5217-21 (2001); Tapper et al., Proc Natl Acad Sci USA102(33):11835-11839 (2005); Maniatis et al., Proc Natl Acad Sci USA99:2228-2233 (2002)).

Thus, in some embodiments, the methods include analysis of polymorphismsthat are within 1 LDU of a polymorphism described herein. Methods areknown in the art for identifying such polymorphisms; for example, theInternational HapMap Project provides a public database that can beused, see hapmap.org, as well as The International HapMap Consortium,Nature 426:789-796 (2003), and The International HapMap Consortium,Nature 437:1299-1320 (2005). Generally, it will be desirable to use aHapMap constructed using data from individuals who share ethnicity withthe subject, e.g., a HapMap for African Americans would ideally be usedto identify markers within 1 LDU of a marker described herein for use ingenotyping a subject of African American descent.

Exemplary polymorphisms that are within 1 LDU of some of the markersdescribed herein are included in the Examples.

Alternatively, methods described herein can include analysis ofpolymorphisms that are within a value defined by Lewontin's D′ (linkagedisequilibrium parameter, see Lewontin, Genetics 49:49-67 (1964)) of apolymorphism described herein. Results can be obtained, e.g., from online public resources such as HapMap.org. The simple linkagedisequilibrium parameter (D) reflects the degree to which alleles at twoloci (for example two SNPs) occur together more often (positive values)or less often (negative values) than expected in a population asdetermined by the products of their respective allele frequencies. Forany two loci, D can vary in value from −0.25 to +0.25. However, themagnitude of D (Dmax) varies as function of allele frequencies. Tocontrol for this, Lewontin introduced the D′ parameter, which is D/Dmaxand varies in value from −1 (alleles never observed together) to +1(alleles always observed together). Typically, the absolute value of D′(i.e., |D′|) is reported in online databases, because it followsmathematically that positive association for one set of alleles at twoloci corresponds to a negative association of equal magnitude for thereciprocal set. This disequilibrium parameter varies from 0 (noassociation of alleles at the two loci) to 1 (maximal possibleassociation of alleles at the two loci).

Thus, in some embodiments, the methods include analysis of polymorphismsthat are within D′>0.5, D′>0.75, or D′=1, for pairwise comparisons, of apolymorphism described herein.

Identification of Additional Markers for Use in the Methods DescribedHerein

In general, genetic markers can be identified using any of a number ofmethods well known in the art. For example, numerous polymorphisms inthe regions described herein are known to exist and are available inpublic databases, which can be searched using methods and algorithmsknown in the art. Alternately, polymorphisms can be identified bysequencing either genomic DNA or cDNA in the region in which it isdesired to find a polymorphism. According to one approach, primers aredesigned to amplify such a region, and DNA from a subject is obtainedand amplified. The DNA is sequenced, and the sequence (referred to as a“subject sequence” or “test sequence”) is compared with a referencesequence, which can represent the “normal” or “wild type” sequence, orthe “affected” sequence. In some embodiments, a reference sequence canbe from, for example, the human draft genome sequence, publiclyavailable in various databases, or a sequence deposited in a databasesuch as GenBank. In some embodiments, the reference sequence is acomposite of ethnically diverse individuals.

In general, if sequencing reveals a difference between the sequencedregion and the reference sequence, a polymorphism has been identified.The fact that a difference in nucleotide sequence is identified at aparticular site that determines that a polymorphism exists at that site.In most instances, particularly in the case of SNPs, only twopolymorphic variants will exist at any location. However, in the case ofSNPs, up to four variants may exist since there are four naturallyoccurring nucleotides in DNA. Other polymorphisms, such as insertionsand deletions, may have more than four alleles.

Other Genetic Markers of Schizophrenia

The methods described herein can also include determining the presenceor absence of other markers known or suspected to be associated with SZ,or with SZ, SD or SPD, e.g., markers outside of a region identifiedherein, see, e.g., Harrison and Owen, Lancet, 361(9355):417-419 (2003),including, for example, markers on chromosome 22 and other chromosomes,e.g., in the region of 22q12.3 (e.g., near D22S283), 22q11.2, 22q11.2,22q11-q13, 1q42.1, 1q42.1, 4p, 18p, 15q15, 14q32.3, 13q34, 13q32, 12q24,11q14-q21, 1q21-q22, 10p15-p13 (e.g., near D105189), 10q22.3, 8p12-21,6q13-q26, 6p22.3, 6p23, 5q11.2-q13.3, and/or 3p25. In some embodiments,the methods include determining the presence or absence of one or moreother markers that are or may be associated with SZ, or with SZ, SD orSPD, e.g., in one or more genes, e.g., ADRA1A (Clark et al., BiolPsychiatry. 58(6):435-9 (2005)); AKT1 (Emamian et al., Nature Genet.36:131-137 (2004)); ALDH3B1 (Sun et al. Sci. China C. Life. Sci.48(3):263-9 (2005)); ARSA (Marcao et al., Mol Genet Metab. 79(4):305-7(2003); ARVCF (Chen et al., Schizophr Res. 72(2-3):275-7 (2005)); BDNF(Neves-Pereira et al., Molec. Psychiat. 10:208-212 (2005)); BZRP(Kurumaji et al., J Neural Transm. 107(4):491-500 (2000)); DAO (Owen etal., Trends Genet. 21(9):518-25 (2005)); DAOA (Owen et al., 2005,supra); CAPON (Brzustowicz et al., Am J Hum Genet. 74(5):1057-63(2004)); CHRNA2 (Blayeri et al., Europ. J. Hum. Genet. 9: 469-472(2001)); COMT (Shifman et al., Am. J. Hum. Genet. 71:1296-1302 (2002));CPLX2 (Lee et al., Behav Brain Funct. 1:15 (2005)); DGCR8 (Jacquet etal., Hum Mol Genet. 11(19):2243-9 (2002)); DISC1 (Owen et al., 2005,supra; see, e.g., the D1S2709 marker (Ekelend et al., Hum. Molec. Genet.10:1611-1617 (2001), HEP3 haplotype, Hennah et al., Hum. Molec. Genet.12: 3151-3159 (2003), and Leu607Pro, Hodgkinson et al., Am. J. Hum.Genet. 75:862-872 (2004), Erratum: Am. J. Hum. Genet. 76:196 (2005));DISC2 (Millar et al., Ann Med. 36(5):367-78 (2004)); DPYSL2 (Hong etal., Am J. Med Genet B Neuropsychiatr Genet. 136(1):8-11 (2005)); DRD1(Coon et al., Am. J. Hum. Genet. 52: 327-334 (1993)); DRD2 (Glatt etal., Am. J. Psychiat. 160:469-476 (2003)); DRD3 (Rybakowski et al.,Molec. Psychiat. 6:718-724 (2001)); DTNBP1 (Owen et al., 2005, supra);EPSIN4 (Am J Hum Genet. 76(5):902-7 (2005)); ErbB; EGF (Futamura et al.,Am. J. Hum. Genet. 52: 327-334 (2002)); GABRA1, GABRA2, GABRA6, GABRP(Petryshen et al., Mol Psychiatry. 10(12):1057 (2005)); GFRA1 (Semba etal., Brain Res Mol Brain Res. 124(1):88-95 (2004)); GNB3 (Kunugi et al.,J. Neural Transm. 109(2):213-8 (2002)); GRIK1 (Shibata et al., PsychiatrGenet. 11(3):139-44 (2001)); GRIK2 (Shibata et al., Psychiatry Res.113(1-2):59-67 (2002)); GRIN1 (Qin et al., Eur J Hum Genet. 13(7):807-14(2005)); GRIN2A, GRIN2B (Abdolmaleky et al., Am J Pharmacogenomics.5(3):149-60 (2005)); GRIN2D (Makino et al., Psychiatr Genet.15(3):215-21 (2005)); GRM3 (Egan et al., Proc Natl Acad Sci USA.101(34):12604-9 (2004)); GRM4 (Ohtsuki et al., Psychiatr Genet.11(2):79-83 (2001)); G30/G72 (Schulze et al., Am J Psychiatry.162(11):2101-8 (2005)); HTR2A (Baritaki et al., Eur J Hum Genet.12(7):535-41 (2004)); HLA-DRB1 (Schwab et al., Am J Med Genet.114(3):315-20 (2002)); HLA-BRB3 (Yu et al., Zhonghua Liu Xing Bing XueZa Zhi. 24(9):815-8 (2003)); IL2RB (Schwab et al., Am J Med Genet.60(5):436-43 (1995)); KCNN3 (Ujike et al., Psychiatry Res. 101(3):203-7(2001)); KIF13A (Jamain et al., Genomics. 74(1):36-44 (2001)); KPNA3(Wei and Hemmings, Neurosci Res. 52(4):342-6 (2005)); LGI1 (Fallin etal. A J Hum Genet. 77:918-36 (2005)); MAG (Wan et al., Neurosci Lett.388(3):126-31 (2005)); MLC1 (Verma et al., Biol Psychiatry. 58(1):16-22(2005)); MTHFR (Lewis et al., Am. J. Med. Genet. (Neuropsychiat. Genet.)135B:2-4 (2005)); NOS1 (Liou et al., Schizophr Res. 65(1):57-9 (2003));NOTCH4 (Wei and Hemmings, (Letter) Nature Genet. 25:376-377 (2000));NRG1 (Owen et al., 2005, supra); NRG3 (Fallin et al. A J Hum Genet.77:918-36 (2005)); PCQAP (Sandhu et al., Psychiatr Genet. 14(3):169-72(2004)); PIK4CA (Saito et al., Am J Med Genet B Neuropsychiatr Genet.116(1):77-83 (2003)); PLA2G4A, PLA2G4C (Yu et al., Prostaglandins LeukotEssent Fatty Acids. 73(5):351-4 (2005)); PPP3CC (Gerber et al., ProcNatl Acad Sci USA. 100(15):8993-8 (2003)); PNOC (Blayeri et al., 2001);PRODH (Chakravarti, Proc. Nat. Acad. Sci. 99:4755-4756 (2002)); QKI(Aberg et al., Am J Med Genet B Neuropsychiatr Genet. 2005 Dec. 9; [Epubahead of print]); RGS4 (Chowdari et al., Hum. Molec. Genet. 11:1373-1380(2002), Erratum: Hum. Molec. Genet. 12:1781 (2003)); RELN (Costa et al.,Mol Interv. 2(1):47-57 (2002)); SCAT (Culkjovic et al., Am J Med Genet.96(6):884-7 (2000)); SLC15A1 (Maheshwari et al., BMC Genomics. 3(1):30(2002)); SLC18A1 (Bly, Schizophr Res. 78(2-3):337-8 (2005)); SNAP29(Saito et al., Mol Psychiatry 6(2):193-201 (2001); Erratum in: MolPsychiatry 6(5):605 (2001); SYNGR1 (Verma et al., Biol Psychiatry.55(2):196-9 (2004)); SYN2 (Chen et al., Bio. Psychiat. 56:177-181(2004)); SYN3 (Porton et al. Biol Psychiatry. 55(2):118-25 (2004));TBP/SCA17 (Chen et al., Schizophr Res. 78(2-3):131-6 (2005)); TPP2(Fallin et al. A J Hum Genet. 77:918-36 (2005)); TRAR4 (Am J Hum Genet.75(4):624-38 (2004)); TRAX (Thomson et al., Mol Psychiatry.10(7):657-68, 616 (2005)); UFD1L (De Luca et al., Am J Med Genet.105(6):529-33 (2001)); YWHAH (Toyooka et al., Am J Med Genet.88(2):164-7 (1999)); ZDHHC8 (Mukai et al., Nature Genet. 36:725-731(2004)); or ZNF74 (Takase et al., Schizophr Res. 52(3):161-5 (2001)).See also, e.g., OMIM entry no. 181500 (SCZD).

Methods of Determining the Presence or Absence of a Haplotype Associatedwith SZ, SPD or SD

The methods described herein include determining the presence or absenceof haplotypes associated with SZ, SPD or SD. In some embodiments, anassociation with SZ is determined by the presence of a shared haplotypebetween the subject and an affected reference individual, e.g., a firstor second-degree relation of the subject, and the absence of thehaplotype in an unaffected reference individual. Thus the methods caninclude obtaining and analyzing a sample from a suitable referenceindividual.

Samples that are suitable for use in the methods described hereincontain genetic material, e.g., genomic DNA (gDNA). Non-limitingexamples of sources of samples include urine, blood, and tissue. Thesample itself will typically consist of nucleated cells (e.g., blood orbuccal cells), tissue, etc., removed from the subject. The subject canbe an adult, child, fetus, or embryo. In some embodiments, the sample isobtained prenatally, either from a fetus or embryo or from the mother(e.g., from fetal or embryonic cells in the maternal circulation).Methods and reagents are known in the art for obtaining, processing, andanalyzing samples. In some embodiments, the sample is obtained with theassistance of a health care provider, e.g., to draw blood. In someembodiments, the sample is obtained without the assistance of a healthcare provider, e.g., where the sample is obtained non-invasively, suchas a sample comprising buccal cells that is obtained using a buccal swabor brush, or a mouthwash sample.

The sample may be further processed before the detecting step. Forexample, DNA in a cell or tissue sample can be separated from othercomponents of the sample. The sample can be concentrated and/or purifiedto isolate DNA. Cells can be harvested from a biological sample usingstandard techniques known in the art. For example, cells can beharvested by centrifuging a cell sample and resuspending the pelletedcells. The cells can be resuspended in a buffered solution such asphosphate-buffered saline (PBS). After centrifuging the cell suspensionto obtain a cell pellet, the cells can be lysed to extract DNA, e.g.,gDNA. See, e.g., Ausubel et al., 2003, supra. All samples obtained froma subject, including those subjected to any sort of further processing,are considered to be obtained from the subject.

The absence or presence of a haplotype associated with SZ, SPD or SD asdescribed herein can be determined using methods known in the art, e.g.,gel electrophoresis, capillary electrophoresis, size exclusionchromatography, sequencing, and/or arrays to detect the presence orabsence of the marker(s) of the haplotype. Amplification of nucleicacids, where desirable, can be accomplished using methods known in theart, e.g., PCR.

Methods of nucleic acid analysis to detect polymorphisms and/orpolymorphic variants include, e.g., microarray analysis. Hybridizationmethods, such as Southern analysis, Northern analysis, or in situhybridizations, can also be used (see Current Protocols in MolecularBiology, Ausubel, F. et al., eds., John Wiley & Sons 2003). To detectmicrodeletions, fluorescence in situ hybridization (FISH) using DNAprobes that are directed to a putatively deleted region in a chromosomecan be used. For example, probes that detect all or a part ofmicrosatellite marker D22s526 can be used to detect microdeletions inthe region that contains that marker.

Other methods include direct manual sequencing (Church and Gilbert,Proc. Natl. Acad. Sci. USA 81:1991-1995 (1988); Sanger et al., Proc.Natl. Acad. Sci. 74:5463-5467 (1977); Beavis et al. U.S. Pat. No.5,288,644); automated fluorescent sequencing; single-strandedconformation polymorphism assays (SSCP); clamped denaturing gelelectrophoresis (CDGE); two-dimensional gel electrophoresis (2DGE orTDGE); conformational sensitive gel electrophoresis (CSGE); denaturinggradient gel electrophoresis (DGGE) (Sheffield et al., Proc. Natl. Acad.Sci. USA 86:232-236 (1989)), mobility shift analysis (Orita et al.,Proc. Natl. Acad. Sci. USA 86:2766-2770 (1989)), restriction enzymeanalysis (Flavell et al., Cell 15:25 (1978); Geever et al., Proc. Natl.Acad. Sci. USA 78:5081 (1981)); quantitative real-time PCR (Raca et al.,Genet Test 8(4):387-94 (2004)); heteroduplex analysis; chemical mismatchcleavage (CMC) (Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401(1985)); RNase protection assays (Myers et al., Science 230:1242(1985)); use of polypeptides that recognize nucleotide mismatches, e.g.,E. coli mutS protein; allele-specific PCR, for example. See, e.g., U.S.Patent Publication No. 2004/0014095, to Gerber et al., which isincorporated herein by reference in its entirety. In some embodiments,the methods described herein include determining the sequence of theentire region of 22q13 described herein as being of interest, e.g.,between and including SNPs rs738596, rs738598, or rs135221 on theproximal end, and rs13884 or rs137853 on the distal end. In someembodiments, the sequence is determined on both strands of DNA.

In order to detect polymorphisms and/or polymorphic variants, it willfrequently be desirable to amplify a portion of genomic DNA (gDNA)encompassing the polymorphic site. Such regions can be amplified andisolated by PCR using oligonucleotide primers designed based on genomicand/or cDNA sequences that flank the site. See e.g., PCR Primer: ALaboratory Manual, Dieffenbach and Dveksler, (Eds.); McPherson et al.,PCR Basics: From Background to Bench (Springer Verlag, 2000); Mattila etal., Nucleic Acids Res., 19:4967 (1991); Eckert et al., PCR Methods andApplications, 1:17 (1991); PCR (eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. No. 4,683,202. Other amplification methods thatmay be employed include the ligase chain reaction (LCR) (Wu and Wallace,Genomics, 4:560 (1989), Landegren et al., Science, 241:1077 (1988),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA,86:1173 (1989)), self-sustained sequence replication (Guatelli et al.,Proc. Nat. Acad. Sci. USA, 87:1874 (1990)), and nucleic acid basedsequence amplification (NASBA). Guidelines for selecting primers for PCRamplification are well known in the art. See, e.g., McPherson et al.,PCR Basics: From Background to Bench, Springer-Verlag, 2000. A varietyof computer programs for designing primers are available, e.g., ‘Oligo’(National Biosciences, Inc, Plymouth Minn.), MacVector (Kodak/IBI), andthe GCG suite of sequence analysis programs (Genetics Computer Group,Madison, Wis. 53711).

In one example, a sample (e.g., a sample comprising genomic DNA), isobtained from a subject. The DNA in the sample is then examined todetermine a haplotype as described herein. The haplotype can bedetermined by any method described herein, e.g., by sequencing or byhybridization of the gene in the genomic DNA, RNA, or cDNA to a nucleicacid probe, e.g., a DNA probe (which includes cDNA and oligonucleotideprobes) or an RNA probe. The nucleic acid probe can be designed tospecifically or preferentially hybridize with a particular polymorphicvariant.

In some embodiments, a peptide nucleic acid (PNA) probe can be usedinstead of a nucleic acid probe in the hybridization methods describedabove. PNA is a DNA mimetic with a peptide-like, inorganic backbone,e.g., N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T orU) attached to the glycine nitrogen via a methylene carbonyl linker(see, e.g., Nielsen et al., Bioconjugate Chemistry, The AmericanChemical Society, 5:1 (1994)). The PNA probe can be designed tospecifically hybridize to a nucleic acid comprising a polymorphicvariant conferring susceptibility to or indicative of the presence ofSZ.

In some embodiments, restriction digest analysis can be used to detectthe existence of a polymorphic variant of a polymorphism, if alternatepolymorphic variants of the polymorphism result in the creation orelimination of a restriction site. A sample containing genomic DNA isobtained from the individual. Polymerase chain reaction (PCR) can beused to amplify a region comprising the polymorphic site, andrestriction fragment length polymorphism analysis is conducted (seeAusubel et al., Current Protocols in Molecular Biology, supra). Thedigestion pattern of the relevant DNA fragment indicates the presence orabsence of a particular polymorphic variant of the polymorphism and istherefore indicative of the presence or absence of susceptibility to SZ.

Sequence analysis can also be used to detect specific polymorphicvariants. A sample comprising DNA or RNA is obtained from the subject.PCR or other appropriate methods can be used to amplify a portionencompassing the polymorphic site, if desired. The sequence is thenascertained, using any standard method, and the presence of apolymorphic variant is determined.

Allele-specific oligonucleotides can also be used to detect the presenceof a polymorphic variant, e.g., through the use of dot-blothybridization of amplified oligonucleotides with allele-specificoligonucleotide (ASO) probes (see, for example, Saiki et al., Nature(London) 324:163-166 (1986)). An “allele-specific oligonucleotide” (alsoreferred to herein as an “allele-specific oligonucleotide probe”) istypically an oligonucleotide of approximately 10-50 base pairs,preferably approximately 15-30 base pairs, that specifically hybridizesto a nucleic acid region that contains a polymorphism. Anallele-specific oligonucleotide probe that is specific for particular apolymorphism can be prepared using standard methods (see Ausubel et al.,Current Protocols in Molecular Biology, supra).

Generally, to determine which of multiple polymorphic variants ispresent in a subject, a sample comprising DNA is obtained from theindividual. PCR can be used to amplify a portion encompassing thepolymorphic site. DNA containing the amplified portion may bedot-blotted, using standard methods (see Ausubel et al., CurrentProtocols in Molecular Biology, supra), and the blot contacted with theoligonucleotide probe. The presence of specific hybridization of theprobe to the DNA is then detected. Specific hybridization of anallele-specific oligonucleotide probe (specific for a polymorphicvariant indicative of susceptibility to SZ) to DNA from the subject isindicative of susceptibility to SZ.

In some embodiments, fluorescence polarization template-directeddye-terminator incorporation (FP-TDI) is used to determine which ofmultiple polymorphic variants of a polymorphism is present in a subject(Chen et al., (1999) Genome Research, 9(5):492-498). Rather thaninvolving use of allele-specific probes or primers, this method employsprimers that terminate adjacent to a polymorphic site, so that extensionof the primer by a single nucleotide results in incorporation of anucleotide complementary to the polymorphic variant at the polymorphicsite.

Real-time pyrophosphate DNA sequencing is yet another approach todetection of polymorphisms and polymorphic variants (Alderborn et al.,(2000) Genome Research, 10(8):1249-1258). Additional methods include,for example, PCR amplification in combination with denaturing highperformance liquid chromatography (dHPLC) (Underhill, P. A., et al.,Genome Research, Vol. 7, No. 10, pp. 996-1005, 1997).

The methods can include determining the genotype of a subject withrespect to both copies of the polymorphic site present in the genome.For example, the complete genotype may be characterized as −/−, as −/+,or as +/+, where a minus sign indicates the presence of the reference orwild type sequence at the polymorphic site, and the plus sign indicatesthe presence of a polymorphic variant other than the reference sequence.If multiple polymorphic variants exist at a site, this can beappropriately indicated by specifying which ones are present in thesubject. Any of the detection means described herein can be used todetermine the genotype of a subject with respect to one or both copiesof the polymorphism present in the subject's genome.

In some embodiments, it is desirable to employ methods that can detectthe presence of multiple polymorphisms (e.g., polymorphic variants at aplurality of polymorphic sites) in parallel or substantiallysimultaneously. Oligonucleotide arrays represent one suitable means fordoing so. Other methods, including methods in which reactions (e.g.,amplification, hybridization) are performed in individual vessels, e.g.,within individual wells of a multi-well plate or other vessel may alsobe performed so as to detect the presence of multiple polymorphicvariants (e.g., polymorphic variants at a plurality of polymorphicsites) in parallel or substantially simultaneously according to certainembodiments of the invention.

Probes

Nucleic acid probes can be used to detect and/or quantify the presenceof a particular target nucleic acid sequence within a sample of nucleicacid sequences, e.g., as hybridization probes, or to amplify aparticular target sequence within a sample, e.g., as a primer. Probeshave a complimentary nucleic acid sequence that selectively hybridizesto the target nucleic acid sequence. In order for a probe to hybridizeto a target sequence, the hybridization probe must have sufficientidentity with the target sequence, i.e., at least 70%, e.g., 80%, 90%,95%, 98% or more identity to the target sequence. The probe sequencemust also be sufficiently long so that the probe exhibits selectivityfor the target sequence over non-target sequences. For example, theprobe will be at least 20, e.g., 25, 30, 35, 50, 100, 200, 300, 400,500, 600, 700, 800, 900 or more, nucleotides in length. In someembodiments, the probes are not more than 30, 50, 100, 200, 300, 500,750, or 1000 nucleotides in length. Probes are typically about 20 toabout 1×10⁶ nucleotides in length. Probes include primers, whichgenerally refers to a single-stranded oligonucleotide probe that can actas a point of initiation of template-directed DNA synthesis usingmethods such as PCR (polymerase chain reaction), LCR (ligase chainreaction), etc., for amplification of a target sequence.

In some embodiments, the probe is a test probe, e.g., a probe that canbe used to detect polymorphisms in a region described herein, e.g.,polymorphisms as described herein, including D22S526, D22S1749E, and/orother polymorphisms of the Sult4a1 gene lying between SNP markersrs138060 and rs138110. In some embodiments, the probe can hybridize to atarget sequence within a region delimited by SNP rs738596 and SNPrs743615 (described on the internet at ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=738596 andncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=743615, respectively).

In some embodiments, the probe can bind to another marker sequenceassociated with SZ, SPD or SD, as described herein.

Control probes can also be used. For example, a probe that binds a lessvariable sequence, e.g., repetitive DNA associated with a centromere ofa chromosome, can be used as a control. Probes that hybridize withvarious centromeric DNA and locus-specific DNA are availablecommercially, for example, from Vysis, Inc. (Downers Grove, Ill.),Molecular Probes, Inc. (Eugene, Oreg.), or from Cytocell (Oxfordshire,UK). Probe sets are available commercially, e.g., from AppliedBiosystems, e.g., the Assays-on-Demand SNP kits. Alternatively, probescan be synthesized, e.g., chemically or in vitro, or made fromchromosomal or genomic DNA through standard techniques. For example,sources of DNA that can be used include genomic DNA, cloned DNAsequences, somatic cell hybrids that contain one, or a part of one,human chromosome along with the normal chromosome complement of thehost, and chromosomes purified by flow cytometry or microdissection. Theregion of interest can be isolated through cloning, or by site-specificamplification via the polymerase chain reaction (PCR). See, for example,Nath and Johnson, Biotechnic. Histochem., 1998, 73(1):6-22, Wheeless etal., Cytometry 1994, 17:319-326, and U.S. Pat. No. 5,491,224.

In some embodiments, the probes are labeled, e.g., by direct labeling,with a fluorophore, an organic molecule that fluoresces after absorbinglight of lower wavelength/higher energy. A directly labeled fluorophoreallows the probe to be visualized without a secondary detectionmolecule. After covalently attaching a fluorophore to a nucleotide, thenucleotide can be directly incorporated into the probe with standardtechniques such as nick translation, random priming, and PCR labeling.Alternatively, deoxycytidine nucleotides within the probe can betransaminated with a linker. The fluorophore then is covalently attachedto the transaminated deoxycytidine nucleotides. See, e.g., U.S. Pat. No.5,491,224.

Fluorophores of different colors can be chosen such that each probe in aset can be distinctly visualized. For example, a combination of thefollowing fluorophores can be used: 7-amino-4-methylcoumarin-3-aceticacid (AMCA), Texas Red™ (Molecular Probes, Inc., Eugene, Oreg.), 5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC),7-diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5-(and-6)-isothiocyanate, 5-(and -6)-carboxytetramethylrhodamine,7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-3-indacenepropionic acid, eosin-5-isothiocyanate,erythrosin-5-isothiocyanate, and Cascade™ blue acetylazide (MolecularProbes, Inc., Eugene, Oreg.). Fluorescently labeled probes can be viewedwith a fluorescence microscope and an appropriate filter for eachfluorophore, or by using dual or triple band-pass filter sets to observemultiple fluorophores. See, for example, U.S. Pat. No. 5,776,688.Alternatively, techniques such as flow cytometry can be used to examinethe hybridization pattern of the probes. Fluorescence-based arrays arealso known in the art.

In other embodiments, the probes can be indirectly labeled with, e.g.,biotin or digoxygenin, or labeled with radioactive isotopes such as ³²Pand ³H. For example, a probe indirectly labeled with biotin can bedetected by avidin conjugated to a detectable marker. For example,avidin can be conjugated to an enzymatic marker such as alkalinephosphatase or horseradish peroxidase. Enzymatic markers can be detectedin standard colorimetric reactions using a substrate and/or a catalystfor the enzyme. Catalysts for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

Oligonucleotide probes that exhibit differential or selective binding topolymorphic sites may readily be designed by one of ordinary skill inthe art. For example, an oligonucleotide that is perfectly complementaryto a sequence that encompasses a polymorphic site (i.e., a sequence thatincludes the polymorphic site, within it or at one end) will generallyhybridize preferentially to a nucleic acid comprising that sequence, asopposed to a nucleic acid comprising an alternate polymorphic variant.

Arrays and Uses Thereof

In another aspect, the invention features arrays that include asubstrate having a plurality of addressable areas, and methods of usingthem. At least one area of the plurality includes a nucleic acid probethat binds specifically to a sequence in chromosome 22q13, and can beused to detect the absence or presence of a polymorphism, e.g., one ormore SNPs, microsatellites, minisatellites, or indels, as describedherein, to determine a haplotype in this region. For example, the arraycan include one or more nucleic acid probes that can be used to detect apolymorphism listed in table 4, 6, 7, 8, or 9. In some embodiments, thearray further includes at least one area that includes a nucleic acidprobe that can be used to specifically detect another marker associatedwith SZ, SPD or SD, as described herein. The substrate can be, e.g., atwo-dimensional substrate known in the art such as a glass slide, awafer (e.g., silica or plastic), a mass spectroscopy plate, or athree-dimensional substrate such as a gel pad. In some embodiments, theprobes are nucleic acid capture probes.

Methods for generating arrays are known in the art and include, e.g.,photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854;5,510,270; and 5,527,681), mechanical methods (e.g., directed-flowmethods as described in U.S. Pat. No. 5,384,261), pin-based methods(e.g., as described in U.S. Pat. No. 5,288,514), and bead-basedtechniques (e.g., as described in PCT US/93/04145). The array typicallyincludes oligonucleotide probes capable of specifically hybridizing todifferent polymorphic variants. According to the method, a nucleic acidof interest, e.g., a nucleic acid encompassing a polymorphic site,(which is typically amplified) is hybridized with the array and scanned.Hybridization and scanning are generally carried out according tostandard methods. See, e.g., Published PCT Application Nos. WO 92/10092and WO 95/11995, and U.S. Pat. No. 5,424,186. After hybridization andwashing, the array is scanned to determine the position on the array towhich the nucleic acid hybridizes. The hybridization data obtained fromthe scan is typically in the form of fluorescence intensities as afunction of location on the array.

Arrays can include multiple detection blocks (i.e., multiple groups ofprobes designed for detection of particular polymorphisms). Such arrayscan be used to analyze multiple different polymorphisms. Detectionblocks may be grouped within a single array or in multiple, separatearrays so that varying conditions (e.g., conditions optimized forparticular polymorphisms) may be used during the hybridization. Forexample, it may be desirable to provide for the detection of thosepolymorphisms that fall within G-C rich stretches of a genomic sequence,separately from those falling in A-T rich segments. Additionaldescription of use of oligonucleotide arrays for detection ofpolymorphisms can be found, for example, in U.S. Pat. Nos. 5,858,659 and5,837,832. In addition to oligonucleotide arrays, cDNA arrays may beused similarly in certain embodiments of the invention.

The methods described herein can include providing an array as describedherein; contacting the array with a sample, e.g., a portion of genomicDNA that includes at least a portion of human chromosome 22, e.g., aregion between SNP rs738596 and SNP rs743615, and, optionally, adifferent portion of genomic DNA, e.g., a portion that includes adifferent portion of human chromosome 22 or another chromosome, e.g.,including another region associated with SZ, SPD or SD., and detectingbinding of a nucleic acid from the sample to the array. Optionally, themethod includes amplifying nucleic acid from the sample, e.g., genomicDNA that includes a portion of human chromosome 22q13 described herein,and, optionally, a region that includes another region associated withSZ, SPD, or SD, prior to or during contact with the array.

In some aspects, the methods described herein can include using an arraythat can ascertain differential expression patterns or copy numbers ofone or more genes in samples from normal and affected individuals. Forexample, arrays of probes to a marker described herein can be used tomeasure polymorphisms between DNA from a subject having SZ, SPD, or SD,and control DNA, e.g., DNA obtained from an individual that does nothave SZ, SPD, or SD, and has no risk factors for SZ, SPD, or SD. Sincethe clones on the array contain sequence tags, their positions on thearray are accurately known relative to the genomic sequence. Differenthybridization patterns between DNA from an individual afflicted with SZand DNA from a normal individual at areas in the array corresponding tomarkers in human chromosome 22q13 described herein, and, optionally, oneor more other regions associated with SZ, SPD, or SD, are indicative ofa risk of SZ. Methods for array production, hybridization, and analysisare described, e.g., in Snijders et al., (2001) Nat. Genetics29:263-264; Klein et al., (1999) Proc. Natl. Acad. Sci. U.S.A.96:4494-4499; Albertson et al., (2003) Breast Cancer Research andTreatment 78:289-298; and Snijders et al. “BAC microarray basedcomparative genomic hybridization.” In: Zhao et al. (eds), BacterialArtificial Chromosomes: Methods and Protocols, Methods in MolecularBiology, Humana Press, 2002.

In another aspect, the invention features methods of determining theabsence or presence of a haplotype associated with SZ as describedherein, using an array described above. The methods include providing atwo dimensional array having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other addressof the plurality having a unique nucleic acid capture probe, contactingthe array with a first sample from a test subject who is suspected ofhaving or being at risk for SZ, and comparing the binding of the firstsample with one or more references, e.g., binding of a sample from asubject who is known to have SZ, SPD, or SD, and/or binding of a samplefrom a subject who is unaffected, e.g., a control sample from a subjectwho neither has, nor has any risk factors for SZ, SPD, or SD. In someembodiments, the methods include contacting the array with a secondsample from a subject who has SZ, SPD or SD; and comparing the bindingof the first sample with the binding of the second sample. In someembodiments, the methods include contacting the array with a thirdsample from a cell or subject that does not have SZ and is not at riskfor SZ; and comparing the binding of the first sample with the bindingof the third sample. In some embodiments, the second and third samplesare from first or second-degree relatives of the test subject. Binding,e.g., in the case of a nucleic acid hybridization, with a capture probeat an address of the plurality, can be detected by any method known inthe art, e.g., by detection of a signal generated from a label attachedto the nucleic acid.

Schizophrenia, Schizotypal Personality Disorder, and SchizoaffectiveDisorder

The methods described herein can be used to determine an individual'srisk of developing schizophrenia (SZ), schizotypal personality disorder(SPD), and/or a schizoaffective disorder (SD).

Schizophrenia (SZ)

SZ is considered a clinical syndrome, and is probably a constellation ofseveral pathologies. Substantial heterogeneity is seen between cases,which is thought to reflect multiple overlapping etiologic factors,including both genetic and environmental contributions. A diagnosis ofSZ is typically indicated by chronic psychotic symptoms, e.g.,hallucinations and delusions. Disorganization of thought and behaviorare common and are considered distinguishing factors in the diagnosis ofSZ. Patients typically have some subtle impairments in cognition.Reduced emotional experience and expression, low drive, and impairedspeech are observed in a subgroup of patients. Cognitive, emotional andsocial impairments often appear early in life, while the psychoticsymptoms typically manifest in late adolescence or early adulthood inmen, a little later in women.

A diagnosis of SZ can be made according to the criteria reported in theDiagnostic and Statistical Manual of Mental Disorders, Fourth Edition,Text Revision, American Psychiatric Association, 2000, (referred toherein as DSM-IV) as follows:

Diagnostic Criteria for SZ

All six criteria must be met for a diagnosis of SZ.

A. Characteristic symptoms: Two (or more) of the following, each presentfor a significant portion of time during a one month period (or less ifsuccessfully treated):

-   -   (1) delusions    -   (2) hallucinations    -   (3) disorganized speech (e.g., frequent derailment or        incoherence)    -   (4) grossly disorganized or catatonic behavior    -   (5) negative symptoms, e.g., affective flattening, alogia, or        avolition

Only one criterion A symptom is required if delusions are bizarre orhallucinations consist of a voice keeping up a running commentary on theperson's behavior or thoughts, or two or more voices conversing witheach other.

B. Social/occupational dysfunction: For a significant portion of thetime since the onset of the disturbance, one or more major areas offunctioning such as work, interpersonal relations, or self-care aremarkedly below the level achieved prior to the onset (or when the onsetis in childhood or adolescence, failure to achieve expected level ofinterpersonal, academic, or occupational achievement).

C. Duration: Continuous signs of the disturbance persist for at least 6months. This 6-month period must include at least 1 month of symptoms(or less if successfully treated) that meet Criterion A (i.e.,active-phase symptoms) and may include periods of prodromal or residualsymptoms. During these prodromal or residual periods, the signs of thedisturbance may be manifested by only negative symptoms or two or moresymptoms listed in Criterion A present in an attenuated form (e.g., oddbeliefs, unusual perceptual experiences).

D. Schizoaffective and Mood Disorder Exclusion: Schizoaffective Disorderand Mood Disorder With Psychotic Features have been ruled out becauseeither (1) no major depressive, manic, or mixed episodes have occurredconcurrently with the active-phase symptoms; or (2) if mood episodeshave occurred during active-phase symptoms, their total duration hasbeen brief relative to the duration of the active and residual periods.

E. Substance/General Medical Condition Exclusion: The disturbance is notdue to the direct physiological effects of a substance (e.g., a drug ofabuse, a medication) or a general medical condition.

F. Relationship to a Pervasive Developmental Disorder: If the patienthas a history of Autistic Disorder or another Pervasive DevelopmentalDisorder, the additional diagnosis of SZ is made only if prominentdelusions or hallucinations are also present for at least a month (orless if successfully treated).

Schizoaffective Disorder (SD)

SD is characterized by the presence of affective (depressive or manic)symptoms and schizophrenic symptoms within the same, uninterruptedepisode of illness.

Diagnostic Criteria for Schizoaffective Disorder

The DSM-IV Criteria for a diagnosis of schizoaffective disorder is asfollows:

An uninterrupted period of illness during which, at some time, there iseither (1) a Major Depressive Episode (which must include depressedmood), (2) a Manic Episode, or (3) a Mixed Episode, concurrent withsymptoms that meet (4) Criterion A for SZ, above.

A. Criteria for Major Depressive Episode

At least five of the following symptoms must be present during the same2-week period and represent a change from previous functioning; at leastone of the symptoms is either (1) depressed mood or (2) loss of interestor pleasure.

(1) depressed mood most of the day, nearly every day, as indicated byeither subjective report (e.g., feels sad or empty) or observation madeby others (e.g., appears tearful). In children and adolescents, this canbe an irritable mood.

(2) markedly diminished interest or pleasure in all, or almost all,activities most of the day, nearly every day (as indicated by eithersubjective account or observation made by others)

(3) significant weight loss when not dieting or weight gain (e.g., achange of more than 5% of body weight in a month), or decrease orincrease in appetite nearly every day. (In children, failure to makeexpected weight gains is considered).

(4) insomnia or hypersomnia nearly every day

(5) psychomotor agitation or retardation nearly every day (observable byothers, not merely subjective feelings of restlessness or being sloweddown)

(6) fatigue or loss of energy nearly every day

(7) feelings of worthlessness or excessive or inappropriate guilt (whichmay be delusional) nearly every day (not merely self-reproach or guiltabout being sick)

(8) diminished ability to think or concentrate, or indecisiveness,nearly every day (either by subjective account or as observed by others)

(9) recurrent thoughts of death (not just fear of dying), recurrentsuicidal ideation without a specific plan, or a suicide attempt or aspecific plan for committing suicide

In addition, the symptoms do not meet criteria for a Mixed Episode. Thesymptoms cause clinically significant distress or impairment in social,occupational, or other important areas of functioning. The symptoms arenot due to the direct physiological effects of a substance (e.g., a drugof abuse, a medication) or a general medical condition (e.g.,hypothyroidism).

The symptoms are not better accounted for by Bereavement, i.e., afterthe loss of a loved one, the symptoms persist for longer than 2 months,or are characterized by marked functional impairment, morbidpreoccupation with worthlessness, suicidal ideation, psychotic symptoms,or psychomotor retardation.

B. Criteria for Manic Episode

A manic episode is a distinct period of abnormally and persistentlyelevated, expansive, or irritable mood, lasting at least one week (orany duration, if hospitalization is necessary).

During the period of mood disturbance, three (or more) of the followingsymptoms have persisted (four if the mood is only irritable) and havebeen present to a significant degree:

-   -   (1) inflated self-esteem or grandiosity    -   (2) decreased need for sleep (e.g., feels rested after only 3        hours of sleep)    -   (3) more talkative than usual or pressure to keep talking    -   (4) flight of ideas or subjective experience that thoughts are        racing    -   (5) distractibility (i.e., attention too easily drawn to        unimportant or irrelevant external stimuli)    -   (6) increase in goal-directed activity (either socially, at work        or school, or sexually) or psychomotor agitation    -   (7) excessive involvement in pleasurable activities that have a        high potential for painful consequences (e.g., engaging in        unrestrained buying sprees, sexual indiscretions, or foolish        business investments)

The symptoms do not meet criteria for a Mixed Episode. The mooddisturbance is sufficiently severe to cause marked impairment inoccupational functioning or in usual social activities or relationshipswith others, or to necessitate hospitalization to prevent harm to selfor others, or there are psychotic features. The symptoms are not due tothe direct physiological effects of a substance (e.g., a drug of abuse,a medication, or other treatment) or a general medical condition (e.g.,hyperthyroidism).

C. Criteria for Mixed Episode

A mixed episode occurs when the criteria are met both for a ManicEpisode and for a Major Depressive Episode (except for duration) nearlyevery day during at least a 1-week period. The mood disturbance issufficiently severe to cause marked impairment in occupationalfunctioning or in usual social activities or relationships with others,or to necessitate hospitalization to prevent harm to self or others, orthere are psychotic features.

The symptoms are not due to the direct physiological effects of asubstance (e.g., a drug of abuse, a medication, or other treatment) or ageneral medical condition (e.g., hyperthyroidism).

D. Criterion A of SZ

See above.

E. Types of SD

The type of SD may be may be specifiable, as either Bipolar Type, if thedisturbance includes a Manic or a Mixed Episode (or a Manic or a MixedEpisode and Major Depressive Episodes), or Depressive Type, if thedisturbance only includes Major Depressive Episodes.

F. Associated Features

Features associated with SD include Learning Problems, Hypoactivity,Psychotic, Euphoric Mood, Depressed Mood, Somatic/Sexual Dysfunction,Hyperactivity, Guilt/Obsession, Odd/Eccentric/Suspicious Personality,Anxious/Fearful/Dependent Personality, and Dramatic/Erratic/AntisocialPersonality.

Schizotvpal Personality Disorder (SPD)

Diagnostic Criteria for SPD

A diagnosis of SPD under the criteria of the DSM-IV is generally basedon a pervasive pattern of social and interpersonal deficits marked byacute discomfort with, and reduced capacity for, close relationships aswell as by cognitive or perceptual distortions and eccentricities ofbehavior, beginning by early adulthood and present in a variety ofcontexts, as indicated by five (or more) of the following:

(1) ideas of reference (excluding delusions of reference)

(2) odd beliefs or magical thinking that influences behavior and is

(3) inconsistent with subcultural norms (e.g., superstitiousness, beliefin clairvoyance, telepathy, or “sixth sense;” in children andadolescents, bizarre fantasies or preoccupations)

(4) unusual perceptual experiences, including bodily illusions

(5) odd thinking and speech (e.g., vague, circumstantial, metaphorical,overelaborate, or stereotyped)

(6) suspiciousness or paranoid ideation

(7) inappropriate or constricted affect

(8) behavior or appearance that is odd, eccentric, or peculiar

(9) lack of close friends or confidants other than first-degreerelatives

(10) excessive social anxiety that does not diminish with familiarityand tends to be associated with paranoid fears rather than negativejudgments about self

SPD is diagnosed if the symptoms do not occur exclusively during thecourse of SZ, a Mood Disorder With Psychotic Features, another PsychoticDisorder, or a Pervasive Developmental Disorder, and the disturbance isnot due to the direct physiological effects of a substance (e.g., a drugof abuse, a medication) or a general medical condition.

Associated features of SPD include Depressed Mood andOdd/Eccentric/Suspicious Personality.

Endophenotypes in SZ

A number of endophenotypes, i.e., intermediate phenotypes, that may moreclosely reflect biological mechanisms behind SZ, have been suggested,such as prepulse inhibition, structural abnormalities evident in MRIscans, specific domains of cognition (e.g., executive function), finemotor performance, working memory, etc.

Endophenotypes can also include clinical manifestations such ashallucinations, paranoia, mania, depression, obsessive-compulsivesymptoms, etc., as well as response or lack of response to drugs andcomorbidity for substance and alcohol abuse.

See, e.g., Kendler et al., Am J Psychiatry 152(5):749-54 (1995);Gottesman and Gould, Am J Psychiatry 160(4):636-45 (2003); Cadenhead,Psychiatric Clinics of North America. 25(4):837-53 (2002); Gottesman andGould, American Journal of Psychiatry. 160(4):636-45 (2003); Heinrichs,Neuroscience & Biobehavioral Reviews. 28(4):379-94 (2004); and Zobel andMaier, Nervenarzt. 75(3):205-14 (2004).

There is now evidence that some candidate genes that were identifiedusing DSM-IV type categorical definitions for “affected” individuals mayinfluence specific endophenotypes, see, e.g., Baker et al., BiolPsychiatry 58(1):23-31 (2005); Cannon et al., Arch Gen Psychiatry62(11):1205-13 (2005); Gothelf et al., Nat Neurosci 8(11):1500-2 (2005);Hallmayer et al., Am J Hum Genet 77(3):468-76 (2005); Callicott et al.,Proc Natl Acad Sci USA 102(24):8627-32 (2005); Gornick et al., J AutismDev Disord 1-8 (2005). Thus, the methods described herein can be used toassociate haplotypes of 22q13 with specific endophenotypes.

Current Treatment of SZ, SD, or SPD

Subjects with SZ typically require acute treatment for psychoticexacerbations, and long-term treatment including maintenance andprophylactic strategies to sustain symptom improvement and preventrecurrence of psychosis. Subjects with schizoaffective disorderexperience the symptoms of both SZ and affective disorder (manic and/ordepressive), thus require the specific treatments for each disorder.Subjects with SPD sometimes require medication for acute psychoticepisodes but are often treated using psychosocial methods. The methodsdescribed herein can include the administration of one or more acceptedor experimental treatment modalities to a person identified as at riskof developing SZ, SPD, or a SD, based on the presence of a haplotypeassociated with SZ, SPD, or SD. Currently accepted treatments presentlyinclude both pharmacologic and psychosocial management, and occasionallyelectroconvulsive therapy (ECT).

Standard pharmacologic therapies for SZ and SD include theadministration of one or more antipsychotic medications, which aretypically antagonists acting at postsynaptic D₂ dopamine receptors inthe brain. Antipsychotic medications include conventional, or firstgeneration, antipsychotic agents, which are sometimes referred to asneuroleptics because of their neurologic side effects, and secondgeneration antipsychotic agents, which are less likely to exhibitneuroleptic effects and have been termed atypical antipsychotics.

In some embodiments, the methods described herein include theadministration of one or more antipsychotic medications to a personidentified by a method described herein as being at risk of developingSZ, SPD, or SD. Antipsychotic medications substantially reduce the riskof relapse in the stable phase of illness. In some embodiments, themethods include the administration of a first generation antipsychoticmedication at a dose that is around the “extrapyramidal symptom (EPS)threshold” (i.e., the dose that will induce extrapyramidal side effects,e.g., bradykinesia, rigidity, or dyskinesia, with minimal rigiditydetectable on physical examination, and/or a second-generationantipsychotics at a dose that is therapeutic, yet below the EPSthreshold.

Standard pharmacologic therapies for SD also include the administrationof a combination of antidepressant, and anti-anxiety medication.Suitable antidepressants include serotonergic antidepressants, e.g.,fluoxetine or trazodone. Suitable anxiolytics include benzodiazepines,e.g., lorazepam, clonazepam. Lithium can also be administered. Thus, insome embodiments, the methods can include the administration of one ormore antidepressant and/or anti-anxiety medications to a personidentified as at risk of developing SZ, SPD, or SD.

The methods can also include psychosocial and rehabilitationinterventions, e.g., interventions that are generally accepted astherapeutically beneficial, e.g., cognitive-behavioral therapy fortreatment-resistant positive psychotic symptoms; supportive,problem-solving, educationally oriented psychotherapy; family therapyand education programs aimed at helping patients and their familiesunderstand the patient's illness, reduce stress, and enhance copingcapabilities; social and living skills training; supported employmentprograms; and/or the provision of supervised residential livingarrangements.

Currently accepted treatments for SZ are described in greater detail inthe Practice Guideline for the Treatment of Patients With SchizophreniaAmerican Psychiatric Association, Second Edition, American PsychiatricAssociation, 2004, which is incorporated herein by reference in itsentirety.

Methods of Determining Treatment Regimens and Methods of Treating SZ,SPD or SD

As described herein, the presence of haplotypes described herein atchromosome 22q13 has been correlated with poor patient prognosis. Thus,the new methods can also include selecting a treatment regimen for asubject determined to be at risk for developing SZ, SPD or SD, basedupon the absence or presence of a haplotype associated with SZ asdescribed herein. The determination of a treatment regimen can also bebased upon the absence or presence of other risk factors associated withSZ, e.g., as described herein. Therefore, the methods of the inventioncan include selecting a treatment regimen for a subject having one ormore risk factors for SZ, and having a haplotype described herein atchromosome 22q13. The methods can also include administering a treatmentregimen to a subject having, or at risk for developing, SZ to therebytreat, prevent or delay further progression of the disease. A treatmentregimen can include the administration of antipsychotic medications to asubject identified as at risk of developing SZ before the onset of anypsychotic episodes.

As used herein, the term “treat” or “treatment” is defined as theapplication or administration of a treatment regimen, e.g., atherapeutic agent or modality, to a subject, e.g., a patient. Thesubject can be a patient having SZ, a symptom of SZ or at risk ofdeveloping (i.e., a predisposition toward) SZ. The treatment can be tocure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate,improve or affect SZ, the symptoms of SZ or the predisposition towardSZ.

The methods of the invention, e.g., methods of determining a treatmentregimen and methods of treatment or prevention of SZ, can furtherinclude the step of monitoring the subject, e.g., for a change (e.g., anincrease or decrease) in one or more of the diagnostic criteria for SZlisted herein, or any other parameter related to clinical outcome. Thesubject can be monitored in one or more of the following periods: priorto beginning of treatment; during the treatment; or after one or moreelements of the treatment have been administered. Monitoring can be usedto evaluate the need for further treatment with the same or a differenttherapeutic agent or modality. Generally, a decrease in one or more ofthe parameters described above is indicative of the improved conditionof the subject, although with red blood cell and platelet levels, anincrease can be associated with the improved condition of the subject.

The methods can be used, e.g., to evaluate the suitability of, or tochoose between alternative treatments, e.g., a particular dosage, modeof delivery, time of delivery, inclusion of adjunctive therapy, e.g.,administration in combination with a second agent, or generally todetermine the subject's probable drug response genotype. In a preferredembodiment, a treatment for SZ can be evaluated by administering thesame treatment or combinations or treatments to a subject having SZ, SPDor SD and a haplotype as described herein at human chromosome 22q13 andto a subject that has SZ but does not have a haplotype as describedherein at human chromosome 22q13. The effects of the treatment orcombination of treatments on each of these subjects can be used todetermine if a treatment or combination of treatments is particularlyeffective on a sub-group of subjects having SZ, SPD or SD. In otherembodiments, various treatments or combinations of treatments can beevaluated by administering two different treatments or combinations oftreatments to at least two different subjects having SZ, SPD or SD and ahaplotype as described herein in human chromosome 22q13. Such methodscan be used to determine if a particular treatment or combination oftreatments is more effective than others in treating this subset of SZ,SPD and/or SD patients.

Various treatment regimens are known for treating SZ, e.g., as describedherein.

Pharmacogenomics

With regards to both prophylactic and therapeutic methods of treatmentof SZ, such treatments may be specifically tailored or modified, basedon knowledge obtained from the field of pharmacogenomics.“Pharmacogenomics,” as used herein, refers to the application ofgenomics technologies such as structural chromosomal analysis, to drugsin clinical development and on the market. See, for example, Eichelbaumet al., Clin. Exp. Pharmacol. Physiol. 23:983-985 (1996) and Linder etal., Clin. Chem. 43:254-266 (1997). Specifically, as used herein, theterm refers the study of how a patient's genes determine his or herresponse to a drug (e.g., a patient's “drug response phenotype,” or“drug response genotype”). Thus, another aspect of the inventionprovides methods for tailoring an individual's prophylactic ortherapeutic treatment according to that individual's drug responsegenotype.

Information generated from pharmacogenomic research using a methoddescribed herein can be used to determine appropriate dosage andtreatment regimens for prophylactic or therapeutic treatment of anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when administering a therapeuticcomposition, e.g., a cytotoxic agent or combination of cytotoxic agents,to a patient, as a means of treating or preventing SZ.

In one embodiment, a physician or clinician may consider applyingknowledge obtained in relevant pharmacogenomics studies, e.g., using amethod described herein, when determining whether to administer apharmaceutical composition, e.g., an antipsychotic agent or acombination of antipsychotic agents, to a subject. In anotherembodiment, a physician or clinician may consider applying suchknowledge when determining the dosage, e.g., amount per treatment orfrequency of treatments, of a treatment, e.g., a antipsychotic agent orcombination of antipsychotic agents, administered to a patient.

As one example, a physician or clinician may determine (or havedetermined, e.g., by a laboratory) the haplotype of a subject atchromosome 22q13, and optionally one or more other markers associatedwith SZ, SPD, or SD, of one or a group of subjects who may beparticipating in a clinical trial, wherein the subjects have SZ, SPD, orSD, and the clinical trial is designed to test the efficacy of apharmaceutical composition, e.g., an antipsychotic or combination ofantipsychotic agents, and wherein the physician or clinician attempts tocorrelate the genotypes of the subjects with their response to thepharmaceutical composition.

As another example, information regarding a haplotype associated with anincreased risk of SZ, SPD or SD, as described herein, can be used tostratify or select a subject population for a clinical trial. Theinformation can, in some embodiments, be used to stratify individualsthat may exhibit a toxic response to a treatment from those that willnot. In other cases, the information can be used to separate those thatwill be non-responders from those who will be responders. The haplotypesdescribed herein can be used in pharmacogenomics-based design and managethe conduct of a clinical trial, e.g., as described in U.S. Pat. Pub.No. 2003/0108938.

Theranostics

Also included herein are compositions and methods for the identificationand treatment of subjects who have an increased risk of SZ, SPD or SD,such that a theranostic approach can be taken to test such individualsto determine the effectiveness of a particular therapeutic intervention(e.g., a pharmaceutical or non-pharmaceutical intervention as describedherein) and to alter the intervention to 1) reduce the risk ofdeveloping adverse outcomes and 2) enhance the effectiveness of theintervention. Thus, in addition to diagnosing or confirming thepredisposition to SZ, SPD or SD, the methods and compositions describedherein also provide a means of optimizing the treatment of a subjecthaving such a disorder. Provided herein is a theranostic approach totreating and preventing SZ, SPD or SD, by integrating diagnostics andtherapeutics to improve the real-time treatment of a subject.Practically, this means creating tests that can identify which patientsare most suited to a particular therapy, and providing feedback on howwell a drug is working to optimize treatment regimens.

Within the clinical trial setting, a theranostic method or compositionof the invention can provide key information to optimize trial design,monitor efficacy, and enhance drug safety. For instance, “trial design”theranostics can be used for patient stratification, determination ofpatient eligibility (inclusion/exclusion), creation of homogeneoustreatment groups, and selection of patient samples that arerepresentative of the general population. Such theranostic tests cantherefore provide the means for patient efficacy enrichment, therebyminimizing the number of individuals needed for trial recruitment.“Efficacy” theranostics are useful for monitoring therapy and assessingefficacy criteria. Finally, “safety” theranostics can be used to preventadverse drug reactions or avoid medication error.

The methods described herein can include retrospective analysis ofclinical trial data as well, both at the subject level and for theentire trial, to detect correlations between a haplotype as describedherein and any measurable or quantifiable parameter relating to theoutcome of the treatment, e.g., efficacy (the results of which may bebinary (i.e., yes and no) as well as along a continuum), side-effectprofile (e.g., weight gain, metabolic dysfunction, lipid dysfunction,movement disorders, or extrapyramidal symptoms), treatment maintenanceand discontinuation rates, return to work status, hospitalizations,suicidality, total healthcare cost, social functioning scales, responseto non-pharmacological treatments, and/or dose response curves. Theresults of these correlations can then be used to influencedecision-making, e.g., regarding treatment or therapeutic strategies,provision of services, and/or payment. For example, a correlationbetween a positive outcome parameter (e.g., high efficacy, low sideeffect profile, high treatment maintenance/low discontinuation rates,good return to work status, low hospitalizations, low suicidality, lowtotal healthcare cost, high social function scale, favorable response tonon-pharmacological treatments, and/or acceptable dose response curves)and a selected haplotype can influence treatment such that the treatmentis recommended or selected for a subject having the selected haplotype.

Kits

Also within the scope of the invention are kits comprising a probe thathybridizes with a region of human chromosome at 22q13 and can be used todetect a polymorphism described herein. The kit can include one or moreother elements including: instructions for use; and other reagents,e.g., a label, or an agent useful for attaching a label to the probe.Instructions for use can include instructions for diagnosticapplications of the probe for assessing risk of SZ in a method describedherein. Other instructions can include instructions for attaching alabel to the probe, instructions for performing in situ analysis withthe probe, and/or instructions for obtaining a sample to be analyzedfrom a subject. As discussed above, the kit can include a label, e.g.,any of the labels described herein. In some embodiments, the kitincludes a labeled probe that hybridizes to a region of human chromosomeat 22q13, e.g., a labeled probe as described herein.

The kit can also include one or more additional probes that hybridize tothe same chromosome, e.g., chromosome 22, or another chromosome orportion thereof that can have an abnormality associated with risk forSZ. For example, the additional probe or probes can be: a probe thathybridizes to human chromosome 22q11-12 or a portion thereof, (e.g., aprobe that detects a sequence associated with SZ in this region ofchromosome 22), or probes that hybridize to all or a portion of 22q12.3(e.g., near D22S283), 22q11.2, 22q11.2, 22q11-q13, 1q42.1, 1q42.1, 18p,15q15, 14q32.3, 13q34, 13q32, 12q24, 11q14-q21, 1q21-q22, 10p15-p13(e.g., near D10S189), 10q22.3, 8p21, 6q13-q26, 6p22.3, 6p23,5q11.2-q13.3, and/or 3p25. A kit that includes additional probes canfurther include labels, e.g., one or more of the same or differentlabels for the probes. In other embodiments, the additional probe orprobes provided with the kit can be a labeled probe or probes. When thekit further includes one or more additional probe or probes, the kit canfurther provide instructions for the use of the additional probe orprobes.

Kits for use in self-testing can also be provided. For example, suchtest kits can include devices and instructions that a subject can use toobtain a sample, e.g., of buccal cells or blood, without the aid of ahealth care provider. For example, buccal cells can be obtained using abuccal swab or brush, or using mouthwash.

Kits as provided herein can also include a mailer, e.g., a postage paidenvelope or mailing pack, that can be used to return the sample foranalysis, e.g., to a laboratory. The kit can include one or morecontainers for the sample, or the sample can be in a standard bloodcollection vial. The kit can also include one or more of an informedconsent form, a test requisition form, and instructions on how to usethe kit in a method described herein. Methods for using such kits arealso included herein. One or more of the forms, e.g., the testrequisition form, and the container holding the sample, can be coded,e.g., with a bar code, for identifying the subject who provided thesample.

Databases

Also provided herein are databases that include a list of polymorphismsas described herein, and wherein the list is largely or entirely limitedto polymorphisms identified as useful in performing genetic diagnosis ofor determination of susceptibility to SZ, SPD or SD as described herein.The list is stored, e.g., on a flat file or computer-readable medium.The databases can further include information regarding one or moresubjects, e.g., whether a subject is affected or unaffected, clinicalinformation such as endophenotype, age of onset of symptoms, anytreatments administered and outcomes (e.g., data relevant topharmacogenomics, diagnostics or theranostics), and other details, e.g.,about the disorder in the subject, or environmental or other geneticfactors. The databases can be used to detect correlations between aparticular haplotype and the information regarding the subject, e.g., todetect correlations between a haplotype and a particular endophenotype,or treatment response.

Transgenic Animals and Cells

Also provided herein are non-human transgenic animals and cells thatharbor one or more polymorphism described herein, e.g., one or morepolymorphisms that constitute a haplotype associated with SZ, SPD, orSD. Such animals and cells are useful for studying the effect of apolymorphism on physiological function, and for identifying and/orevaluating potential therapeutic agents for the treatment of SZ, SPD, orSD, e.g., anti-psychotics.

As used herein, a “transgenic animal” is a non-human animal, preferablya mammal in which one or more of the cells of the animal includes atransgene. Examples of transgenic animals include rodents (e.g., rats ormice), non-human primates, rabbits, sheep, dogs, cows, goats, chickens,amphibians, and the like. A transgene as used herein replicates apolymorphism described herein and is integrated into or occurs in thegenome of the cells, e.g., the cultured cells or the cells of atransgenic animal. As one example, included herein are cells in whichone of the various alleles of the Sult4a1 polymorphism has bere-created, e.g., an allele of D22S1749E. Thus, a transgenic animal orcell can be one in which an endogenous Sult4a1 gene has been altered toinclude an allele of D22S1749E, e.g., an allele that is associated withan increased risk of SZ, SD, or SPD. Methods are known in the art forgenerating such animals and cells. e.g., by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell, e.g., a cell of an animal, e.g., an embryonic cell of ananimal, prior to development of the animal.

A transgenic founder animal can be identified based upon the presence ofa transgene in its genome. A transgenic founder animal can then be usedto breed additional animals carrying the transgene. Moreover, transgenicanimals carrying one transgene protein can further be bred to othertransgenic animals carrying other transgenes. The invention alsoincludes populations of cells from a transgenic animal as describedherein.

Also provided are cells, preferably mammalian cells, e.g., neuronal typecells, in which an endogenous gene has been altered to include apolymorphism as described herein. Techniques such as targeted homologousrecombinations, can be used to insert the heterologous DNA as describedin, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published inMay 16, 1991.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application, are hereby expressly incorporated byreference.

EXAMPLES Example 1 Analysis of Microsatellite Markers in Chromosome 22

Twenty-seven nuclear families, comprising 212 individuals, each havingmultiple affected siblings were provided by the Institutes of MentalHealth (NIMH) Schizophrenia Genetics Initiative. Self-description ofheritage was a follows: African-American, 12 families;European/Mediterranean, 11 families; Hispanic, 2 families; other 2families. DSM-III criteria were compiled for all subjects by researchersat Columbia University, Harvard University and Washington University.Detailed information on ascertainment, diagnosis and informed consenthas been previously provided by these groups (Colinger et al., 1998;Faranoe et al., 1998; Kaufmann et al., Am. J. Genet. 81:282-289 (1998)).Using the DSM-III criteria for SZ, the sample contained 55 affectedsibling pairs, and using a broader disease definition that includeschizotypal personality disorder and schizoaffective disorder, thesample contained 100 affected sibling pairs.

Initially, linkage was analyzed using a set of 14 microsatellitemarkers, which are listed in Table 1. As an example, PCR was performedusing primers for the microsatellite marker D22s1749e. The upstreamprimer sequence was 5′-CAGCCGCACGCCATGGAACTCGAAG-3′(SEQ ID NO:1) and thedownstream primer sequence was 5′-GGCGCCATGACGTCACGCCTGC-3′ (SEQ IDNO:2). Each 10 μl reaction contained a final concentration of 5 ng ofgenomic DNA, 10× buffer (Roche), 0.16 U of AmpliTaq Gold, 2.0 mM MgCl₂,1 mM of each dNTP, 0.33 μM of forward and reverse primers, and 10% DMSO.PCR conditions consisted of an initial enzyme activation at 95° C. for 5min, followed by 35 cycles of 93° C. for 2 min, 92° C. for 1 min, 71° C.for 30 s, and 72° C. for 2 min, and a final incubation at 72° C. for 5min. PCR products were analyzed and fragment size was determined usingthe Biomek CEQ 8000 Analysis System.

TABLE 1 Markers on Human Chromosome 22q Marker^(a) Kosambi cM^(b)Distance Mb^(b,c) D22s311 D22s446 2.6000 20.3437-20.3439 D22s315 11.500024.3404-24.3406 D22s275 22.8000 D22s683 30.2000 34.8384-34.8389 D22s27041.5000 41.3780-41.3782 rs138060 44.4000 42.5477 rs138097 44.467842.5755 D22s1749e 44.4863 42.5831-42.5833 rs138110 44.4897 42.5847D22s274 47.0187 43.5897-43.5899 D22s1149 51.3187 44.9934.44.9935D22s1170 56.7187 46.6712-46.6714 rs738596 59.4417 47.6932 rs73859859.4857 47.7102 D22s1169 59.5187 47.7230-47.7231 rs2073224 59.658747.7490 rs738615 59.6617 47.7495 rs135221 60.1217 47.8325 rs76721960.8517 47.9633 sJCW16 60.8917 47.9709 rs848768 61.7811 48.0389 rs84872862.0582 48.0616 rs2269523 62.3402 48.0898 rs737734 62.4936 48.1051rs136770 62.6528 48.1211 D22s526 63.0417 48.1599-48.1602 rs13447463.7602 48.2321 rs134472 63.7671 48.2328 rs134454 63.8946 48.2455rs135819 64.2029 48.2763 rs763126 64.2360 48.3094 rs916363 64.265048.3384 rs1573726 64.2803 48.3537 rs138817 64.3833 48.4839 rs13884464.4047 48.5053 rs137853 64.6264 48.7459 rs1053744 65.0191 49.1759D22s1744 65.1608 49.3178 D22s1743 65.3608 49.3418 ^(a)Markers are listedfrom 22cen to 22qter ^(b)Brennan et al. Genomics 63, 430-423 (2000)^(c)Ensembl

Simple parametric models did not give significant evidence for linkage,regardless of the mode of inheritance or the degree of penetranceassumed. However, a model assuming genetic heterogeneity resulted inmaximum LOD score of 2.6 at marker D22s270 (θ=0) for SZ and a value of3.6 for a broader definition of disease that included schizotypalpersonality disorder (SPD; FIG. 1). In agreement with the findings ofothers, some evidence for linkage near marker D22s683 was seen using thenarrow definition (Vallada et al., Psychiatr. Genet. 5:127-30 (1995);DeLisi et al., Am. J. Psychiatry 159:803-12 (2002); Takahashi et al.,Am. J. Med. Genet. 120B:11-7 (2003)). Similar peaks at D22s270 resultedfrom nonparametric linkage analysis giving LOD scores of 2.5 and 2.7,for the narrow and broad disease definitions, respectively (FIG. 1).Note that the broader disease definition results both in higher LODscores for D22s270 and an increase in the smaller, more distal peakcentered at sJCW16.

Initial mapping of D22s1749e was performed using the MultiMap program(version 2.40) as described previously (Cox Matise et al., NatureGenetics 6:384-390 (1994); Cox-Matise et al., Multimap, Automatedgenetic linkage mapping, version 2.4. (1996); Brennan et al., Genomics63:430-432 (2000)). TDT analysis was performed using TRANSMIT(version2.5.2) (Clayton, Am. J. Hum. Genet. 65(4):1170-7 (1999)), withrare haplotypes aggregated so as to prevent elevation of X² values thatcan arise due to expectations for rare haplotypes. The resulting globalP values for the X² analyses estimate the significance of thetransmission distribution for all haplotypes combined, with rarehaplotypes being treated as a single haplotype. Similarly, X² values fortransmission of individual genotypes and haplotypes, with one degree offreedom, are determined by TRANSMIT.

Example 2 Identification of Sult4a1 as a Candidate Gene

A search for candidate genes near marker D22s270, performed using publicdatabase resources, identified the sulfotransferase-4A1 gene (Sult4a1),which is located within 1.2 Mb of this microsatellite marker, andencodes a brain-specific sulfotransferase believed to be involved indopamine catabolism (Falany et al., Biochem J. 346:857-64 (2000);Sakakibara et al., Gene 285:39-47 (2002); Liyou et al., J. Histochem.Cytochem. 51:655-64 (2003)).

Alignment of the genomic sequences with several corresponding cDNAsequences (Z97055, AF176342, AF188698, AF251263, AK091700, A1832543)indicated, in all likelihood, that the DNA encoding the 5′non-translated leader region of the Sult4a1 mRNA was polymorphic, havinga varying number of imperfect GCC repeats (primary accession numbersZ97055, AF176342, AF188698, AF251263, AK091700, A1832543). To evaluatethis possibility, a PCR procedure was developed to amplify the genomicregion at the 5′ end of the gene.

Briefly, SNPs were analyzed using Applied Biosystems Assays-on-DemandSNP kits. Each 5 μl reaction contained 2.5 μl of Taq Man polymerase,0.25 μl of 20×SNP assay and 2.25 μl of 10 ng genomic DNA. PCR conditionsconsisted of an initial enzyme activation at 95° C. for 10 min, followedby 40 cycles of 95° C. for 15 sec, and 60° C. for 1 min. PCR productswere analyzed using the ABI Prism 7900HT Sequence Detection System. Theregion is very G-C rich and refractory to amplification. Nonetheless,reproducible amplification of the region was obtained for all families,confirming Mendelian inheritance in all cases.

In this sample of families, seven alleles, with one two five nucleotidesseparating adjacent alleles in the series, were observed. The MultiMapprogram was used to confirm that D22s1749e mapped approximately 10 cMdistal to D22s683. Table 2 lists the location, in mb, of this newmicrosatellite marker and the three nearby SNPs that were used for TDTanalysis.

TABLE 2 Markers Used Marker Location on Chromosome 22 (Mb^(a)) rs13806042.5477 rs138097 42.5755 D22s1749e 42.5831 rs138110 42.5847 ^(a)NCBI:www.ncbi.nlm.nih.gov/SNP/

In this sample of families, seven alleles of marker D22s1749e ranging insize from 198 to 216 nucleotides were observed. (Table 3).

TABLE 3 Observed Alleles of D22s1749e Size (nt) Observed frequency^(a)198 0.0022 202 0.0088 207 0.385 209 0.033 212 0.286 213 0.165 216 0.022^(a)Frequency for the NIMH sample using only those parental genotypesthat were directly observed or that could be unambiguously inferred.

Including this new marker in the linkage analysis did not alter thelocation of the maximum LOD scores, which were still observed at markerD22S270. Owing to the increased information content, the maximum LODvalues increased somewhat. Assuming heterogeneity, a maximum LOD scoreof 2.90 was obtained using DSM IIIR criteria, and a maximum LOD score of3.96 was obtained for a broader disease definition that includedschizotypal personality disorder (FIG. 2). Again, nonparametric linkageanalysis provided suggestive evidence for linkage at the same location,with LOD scores of 2.6 and 2.8 for the narrow broad definitions,respectively (FIG. 1, solid and broken black lines).

The sample was further expanded by adding 17 more families to theoriginal 27 families. Using the D22s1749e marker in linkage analysis forthe pooled sample (using a dominant model assuming geneticheterogeneity, a penetrance of 50% for a heterozygote and a 1% allele) asingle point heterogeneous LOD score of 4.78 was obtained for thecombined sample of 44 families (α=0.7).

Consistent with the initial findings, for the pooled sample, D22S1749Eshowed significant deviation from expectation for transmission toaffected offspring using TRANSMIT (Clayton Am J Hum Genet. 65(4):1170-7(1999) (P=0.015 for SZ, and P=0.006 for the broader definition includingSPD).

Example 3 Identification of Haplotypes Including Markers in Sult4a1

The Sult4a1 candidate gene was further evaluated byTransmission/Disequilibrium Test (TDT) analysis employing the newmicrosatellite marker, along with three SNPs in the gene. Table 4summarizes the results of the TDT analysis for these polymorphisms andhaplotypes involving them. Significant results were seen for D22s1749eand various haplotypes involving D22s1749e and the three SNPs inSult4a1. In most cases, the results were more significant for a narrowdefinition of schizophrenia (SZ), than for broader definitions thatincluded schizotypal personality disorder (SPD) or schizoaffectivedisorder (SD).

TABLE 4 TDT Analysis of Sult4a1 Markers P Value for Disease Definition^(a) Marker(s) df SZ SZ + SPD SZ + SPD + SD D22s1749e 4 ^(b) 0.04 0.050.04 rs138060-rs138097 3 ^(b) 0.04 0.12 0.12 rs138060-D22s1749e 7 ^(b)0.008 0.004 0.17 rs138060-rs138097- 9 ^(c) 0.0014 0.0006 0.0055D22s1749e rs138060-D22s1749e- 10 ^(c)  0.0095 0.0017 0.018 rs138110rs138097-D22s1749e- 7 ^(c) 0.04 0.03 0.05 rs138110 rs138060-rs138097- 11^(c)  0.014 0.0064 0.04 D22s1749e-rs138110 ^(a) SZ = schizophrenia, SPD= schizophrenia + schizotypal personality disorder, SD = schizoaffectivedisorder. ^(b) Global chi square values as determined by Transmit, withhaplotypes having a frequencies of 3% or less aggregated. ^(c) Globalchi square values as determined by Transmit with haplotypes having afrequencies of 14% or less aggregated.

Example 4 Identification of Microsatellite Markers in 22q13 Showing TD

All of the microsatellite polymorphisms listed in Table 1 were testedfor evidence of transmission disequilibrium. Other than D22s1749e, onlyD22s256 showed significant results (Table 5).

D22s256 was evaluated using PCR with the following conditions: δ 95° C.,12 min, 1 cycle; 94° C., 15 sec, 60° C., 15 sec, 72° C., 30 sec, 10cycles; 89° C., 15 sec, 60° C., 15 sec, 72° C., 30 sec, 25 cycles; 72°C., 30 min, 1 cycle. The primers were: Left:5′-AGAGCAAGACTCTGTCTCAACA-3′ (SEQ ID NO:3); Right,5′-TTCTCCTTCACTTTCTGCCATG-3′ (SEQ ID NO:4s). The left primer has a HEXflorescent label at the 5′ end. PCR products were analyzed using an ABIPRISM 377 DNA Sequencer with GeneScan and Genotyper software packages.The expected product size was 250 to 308 nt.

In this sample of families, 16 of the 23 alleles of D22s256, ranging insize from 258 to 305 nt, were observed. Using the narrow DSM-IIIcriteria for SZ provided significant results for this marker (P=0.003).Broader disease definitions including SPD or both SPD andschizoaffective disorder (SD) provided even more striking results(P=0.002 and P=0.00009, respectively).

TABLE 5 TDT Analysis of Marker D22s526 Disease definition Chi Square^(a) P ^(b) SZ 24.97 0.003 SZ + SPD 31.93 0.0002 SZ + SPD + SD 33.950.00009 ^(a) Global chi square values as determined by Transmit, withhaplotypes having a frequencies of 3% or less aggregated. ^(b)Probability with 9 df.

Example 5 Identification of Haplotypes Associated with SZ, SPD, and SD-2and 3 Marker Haplotypes

Tables 6 and 7 list two and three marker haplotypes, respectively, thatshowed highly significant deviations from expected transmissionfrequencies for affected offspring under the broadest diseasedefinition, including SZ, schizoaffective disorder, and schizotypalpersonality disorder. The distances are taken from the NCBI database(SNPdb build 125; Genome Build 35.1, September, 2005).

TABLE 6 TDT: Two marker haplotype p ≦ 0.001 SZ + SD + SPD Marker p valueDistances Mb rs2073224 - D22s526 1.5217E−05 47.7490-48.1602 rs738615-D22s526 1.5217E−05 47.7495-48.1602 rs767219- D22s526 0.001047.9633-48.1602 rs767219 - rs1573726 0.0002 47.9633-48.3537 D22s526 -rs134472 0.0003 48.1602-48.2328 D22s526 - rs763126 3.787E−0748.1602-48.3094 D22s526 - rs1573726 0.0004 48.1602-48.3537 D22s526 -rs138817 8.741E−09 48.1602-48.4839 D22s526 - rs138844 7.976E−1048.1602-48.5053

TABLE 7 TDT: Three marker haplotype p ≦ 0.001 SZ + SD + SPD Markers pvalue Distances Mb rs2073224-rs135221- rs767219 7.4504E−0647.7490-47.9633 rs2073224- rs135221- rs138817 4.0105E−09 47.7490-48.4839rs2073224- rs767219- rs737734 8.3037E−29 47.7490-48.1051 rs2073224-rs767219-rs1573726 4.26677E−05 47.7490-48.3537rs2073224-rs2269523-rs134472 1.9771E−09 47.7490-48.2328 rs135221-rs767219- rs916363 9.7080E−15 47.8325-48.3384 rs135221- rs767219-rs1573726 0.0002 47.8325-48.3537 rs135221- rs848768- rs15737264.7207E−05 47.8325-48.3537 rs135221- rs848768- rs138817 6.6501E−0847.8325-48.4839 rs135221- rs848768-rs1053744 1.1167E−08 47.8325-49.1759rs135221-rs737734-rs134472 9.8432E−06 47.8325-48.2328 rs135221-rs134474-rs1053744 1.6512E−12 47.8325-49.1759 rs135221- rs916363- rs1388178.9512E−242 47.8325-48.4839 rs135221- rs916363- rs1053744 2.2041E−0547.8325-49.1759 rs767219- rs848768- rs2269523 0.0001 47.9633-48.0898rs767219- rs848768- rs737734 9.9720E−06 47.9633-48.1051 rs767219-rs848768- rs1573726 5.1963E−05 47.9633-48.3537 rs767219- rs848768-rs138817 2.5709E−08 47.9633-48.4839 rs767219-rs848728- rs737734 0.000947.9633-48.1051 rs767219-rs848728-rs136770 1.4604E−10 47.9633-48.1211rs767219-rs848728-rs134472 9.8306E−23 47.9633-48.2328 rs767219-rs848728-rs1573726 0.0006 47.9633-48.3537 rs767219- rs2269523-rs737734 4.0063E−1647.9633-48.1051 rs767219- rs2269523- rs1573726 0.0004 47.9633-48.3537rs767219- rs737734-rs916363 3.8423E−06 47.9633-48.3384 rs848768-rs2269523-rs138817 3.3795E−08 48.0389-48.4839 rs136770-rs134474-rs7631263.4059E−05 48.1211-48.3094

Additional haplotypes within this region were also evaluated, and theresults are presented in Table 8. Haplotypes listed in bold show highlysignificant results for the narrowest disease definition of SZ.

TABLE 8 Examples of additional haplotypes p ≦ 0.001 for various diseasedefinitions^(a) Disease definition Single Nucleotide Haplotypes SZ^(b)SZ + SD^(c) SZ + SPD^(d) SZ + SD + SPD^(e) rs1355221-rs1053744 0.13090.0648 0.0003 3.96E−05 rs738596-rs763126 0.082 0.1049 0.4214 1.60E−16rs135221-rs48768-rs1053744 0.0717 0.6809 0.0018 3.26E−27rs135221-rs738615-rs138817 0.0677 0.8333 0.3203 5.47E−11rs136770-rs134474-rs763126 0.0635 0.0792 2.28E−05 3.4E−05rs135221-rs738598-rs2269523 0.0476 0.0021 0.0233 0.0011rs135221-rs916363-rs138817 0.0320 0.0002 0.0214 2.2E−05rs135221-rs763126-rs1573726 0.0154 0.0005 0.0162 0.0017rs848768-rs738598-rs1573726 0.0065 0.0031 0.0109 1.48E−26rs738598-rs2269523-rs1573726 0.0059 0.0015 0.0109 7.51E−17rs738598-rs738615-rs1573726 0.0041 0.0010 0.5106 0.6582rs737734-rs136770-rs763126 0.0010 0.0002 0.0241 0.0126rs738598-rs1573726-rs138844 0.0008 0.0002 0.0003 7.18E−05rs738596-rs738598-rs1573726 0.0007 0.0003 0.0031 0.0023rs738596-rs1573726 0.0004 0.0007 0.0024 0.0040 rs738598-rs1573726 0.00040.0002 0.0109 0.0058 rs135819-rs1573726 0.0004 0.0008 0.0448 0.0467rs738598-rs138844-rs1053744 0.0002 2.39E−05 0.0043 0.0012 rs15737268.06E−05 0.0001 0.0013 0.0015 rs153221-rs763126 4.28E−05 0.0073 0.00210.0310 rs135221-rs2269523-rs737734 9.76E−06 0.1564 0.0001 0.0560rs737734-rs134474-rs134454 5.76E−07 3.85E−05 0.7276 0.5819rs135221-rs848768-rs763126 3.80E−11 0.0273 0.0951 0.1246rs2073224-rs763126-rs138844 4.10E−53 0.0023 0.0061 0.0071rs738615-rs763126-rs138844 4.10E−53 0.0023 0.0061 0.0071 ^(a)Asdetermined by TRANSMIT (rare haplotypes pooled) ^(b)SZ = schizophrenia^(c)SZ + SD = schizophrenia + schizoaffective disorder ^(d)SZ + SPD =schizophrenia + schizotypal personality disorder ^(e)SZ + SD + SPD =schizophrenia + schizoaffective disorder + schizotypal personalitydisorder

Example 6 Identification of Haplotypes Associated with SZ—Alleles ofSult4A

Table 9 summarizes X² tests for specific haplotypes that were determinedto be transferred more frequently or less frequently than expected toaffected offspring using the narrow DSM-III definition of SZ. The 213 ntallele for D22s1749e was transmitted more often than expected, and the207 nt allele less often than expected to affected offspring. None ofthe SNPs, when used alone, showed X² values for transmissiondisequilibrium that were significant at the P<0.01 level. However,several haplotypes involving these SNPs in combination with D22s1749eshowed significant transmission distortion (Table 9).

TABLE 9 TDT Analysis for Specific Haplotypes (P < 0.01) Transmission toAffected Offspring (DSM-III schizophrenia) Higher/Lower Marker(s) ^(a)Haplotype ^(b) than expected χ² (1 df) P D22s1749e 213 higher 7.230.0071 rs138060- D22s1749e A-213 higher 7.89 0.0049 rs138060- D22s1749eC-207 lower 7.58 0.0059 rs138060-rs138097-D22s1749e C-T-207 lower 6.730.0094 rs138060-rs138097-D22s1749e A-T-213 higher 8.02 0.0046rs138060-D22s1749e-rs138110 A-213-G higher 7.66 0.0056rs138097-D22s1749e-rs138110 T-213-G higher 8.01 0.0046rs138060-rs138097- D22s1749e- A-T-213-G higher 7.83 0.0051 rs138110 ^(a)Polymorphisms are listed in proximal to distal order on the chromosome.^(b) Genotypes give the length (nt) for D22s1749e and specificnucleotide descriptions for each SNP, listed in the same order as themarker names.

The 213 nt allele of Sult4a1 appears to be transmitted more often thanexpected to affected children. The 216 nt allele occurred too rarely inthis small sample for the TDT analysis to be statistically valid, buttentatively, it too appears to be preferentially transmitted to affectedoffspring. These alleles are predicted to encode an mRNA with a longer5′ nontranslated leader sequence than the shorter alleles. As onetheory, not meant to be binding, the longer 5′ leader sequences mightlower translatability of the mRNAs and result in lower final levels ofthe Sult4a1 enzyme. At present, the major physiological substrate(s) ofthe Sult4a1 isozyme is unknown, but in vitro, it functions on a varietyof phenolic compounds structurally resembling the catecholamines(Sakakibara et al., Gene 285:39-47 (2002)).

These findings add to a body of results pointing to a role forchromosome 22q in the etiology of SZ. In agreement with the findings ofothers (Vallada et al., Psychiatr. Genet. 5:127-30 (1995); DeLisi etal., Am J Psychiatry 159:803-12 (2002); Takahashi et al., Am. J. Med.Genet. 120B:11-7 (2003)), evidence for linkage near marker D22s683 isseen at about 30 cM on the linkage map, but the highest LOD score wasobtained at 41.5 cM corresponding to marker D22s270. The smaller peak atD22s683 was most prominent with the narrow disease definition, while abroader disease definition results in an additional distal linkage peakcentered at sJCW16.

Based on TDT analysis, both the Sult4a1 candidate gene and the moredistal region of 22q appear to contribute to the genetic predispositionto SZ. In this sample of families, TDT provided suggestive evidence fora role of the Sult4a1 candidate gene located near marker D22s270,representing the major LOD score peak observed in linkage analysis. Incontrast, no evidence of transmission disequilibrium was seen for mostmicrosatellite markers, including D22s683 and D22s270. However, highlysignificant results, particularly for broader disease definitions, wereseen for marker D22s526, which is located within 200 kb of markersJCW16, corresponding to the more distal peak we see in linkageanalysis.

Taken together, these results support a two locus model, involving aproximal locus, perhaps most significant for a narrowly defined SZ and amore distal locus near D22s526, most likely contributing additionally toSPD, SD and other SZ-spectrum disorders. It now seems clear thatsequences within these chromosomal segments contribute to the geneticpredisposition to these disorders.

Example 7 Identification of Haplotypes Associated with SZ—Microdeletionsat D22s526

As described above, numerous two and three SNP haplotypes spanning thedistal region show highly significant distortions in transmission ratiosfor DSM-IIIR diagnosed SZ and broader disease definitions (P<10⁻⁵). Aclose evaluation of the haplotypes revealed particular SNP haplotypesthat are preferentially transmitted. In about half of the NIMH families,these SNP haplotypes occur as part of a larger haplotype involving asmall subset (two to four per population) of the 23 alleles of a highlypolymorphic marker (D22s526).

The D22s526 microsatellite marker was evaluated in 561 unrelatedindividuals from the Louisville Twin Family Study (comprisingapproximately 70% EA, 25% AA and 5% other). As described in Brennan etal., Genomics 63(3):430-2 (2000), a total of 23 alleles of D22s526 wereobserved, ranging in size from 254 nt to 308 nt inclusive. These allelesare numbered from 1 to 23 (smallest to largest) in Table 10.

TABLE 10 Analysis of D22s526 in Control Sample and NIMH SchizophreniaFamilies Allele Expected Observed frequency of Occurrences of allele inObserved frequency of frequency in frequency of apparent homozygotesNIMH sample^(c) (N = 52) apparent homozygotes in Allele controls^(a)homozygotes^(b) in controls (N = 561) [observed frequency] NIMHsample^(d) (N = 26) 1 0.007 <0.1%  0 0 0 2 0.005 <0.1%  0 0 0 3 0.0650.4% 0 2 [0.038] 0 4 0.102  1% 0.4% 3 [0.058] 0 5 0.041 0.2% 0 0 0 60.112 1.3% 0.4% 1 [0.019] 0 7 0.041 0.2% 0 2 [0.038] 0 8 0.107 1.1% 0 6[0.115] 0 9 0.033 0.1% 0.2% 2 [0.038] 3.8% 10 0.149 2.2% 0.7% 9 [0.173]7.7% 11 0.033 0.1% 0.2% 1 [0.019] 0 12 0.102 1.0% 0.2% 9 [0.173] 7.7% 130.080 0.6% 0.7% 1 [0.019] 0 14 0.051 0.2% 0 6 [0.115] 3.8% 15 0.074 0.5%0.5% 0 0 16 0.020 <0.1%  0.2% 0 0 17 0.063 0.4% 0.2% 6 [0.115] 11.5%  180.003 <0.1%  0 1 [0.019] 0 19 0.038 0.1% 0.2% 1 [0.019] 0 20 0.002<0.1%  0 0 0 21 0.018 <0.1%  0 1 [0.019] 0 22 0.009 <0.1%  0 1 [0.019] 023 0.054 0.3% 0.2% 0 0 ^(a)Frequency observed in 561 unrelatedindividuals representing a cross section of the population in theLouisville metropolitan area. The values do not add to 1.00 due torounding. ^(b)Expected frequency of homozygous individuals for anunselected sample given Hardy-Weinberg assumptions. ^(c)Occurrences andempirical frequencies of the alleles in 26 probands from NIHMSchizophrenia Genetics Initiative. ^(d)Observed frequency of apparentlyhomozygous (or hemizygous) individuals in a sample of 26 probands fromNIHM Schizophrenia Genetics Initiative.

An apparent heterozygosity of 97.5% was found in the unselected sampleof 561 unrelated individuals. In other words, one expects only about2.5% of randomly sampled individuals to be homozygous for this marker.By contrast, 9 of 27 (33%) of the NIMH probands (i.e., the individualsfirst identified as affected for each particular family) are apparenthomozygotes (5 of 13 EA; 2 of 12 AA; 1 of 2 “other”; Fisher Exact TestP=2×10⁻⁵; OR15.3; Log odds=2.7).

At least a portion of the apparent homozygosity for this region appearsto be due to microdeletions segregating in some families. Mendelianinheritance patterns for the control sample showed that 4 of the 15apparently homozygous individuals could be hemizygous, because they failto transmit the expected allele to one or more children. Thus, perhapsabout 0.5% of individuals from the unselected population are hemizygousfor D22s526.

A closer look at the NIMH families indicates that microdeletions arelikely. Six of the eight probands with apparent homozygosity for themicrosatellite polymorphism also have an adjacent region of presumptivehemizygosity extending over approximately 200 to 300 kb in one or bothdirections. Furthermore, in AA families in particular, there are fiveadditional probands who appear to carry deletions that do not uncoverthe microsatellite but do uncover nearby extended regions of at least100 to 500 kb, as judged by apparent homozygosity for certain (andvarious) infrequent haplotypes.

DNA from one or both parents and multiple siblings can be used to ruleout most trivial explanations for these results. Loss of homozygosityduring immortalization and propagation of cell lines is unlikely, as thesame presumptive deletion is carried by multiple family members.Consanguinity and resulting extended regions of homozygosity cannotexplain the results either, because other polymorphic markers, even onthe same chromosome, do show extensive homozygosity.

These novel microdeletions may confer significant risk of developingschizophrenia spectrum disorders (SZ, SD, and/or SPD). As one theory,not meant to be limiting, using the 22q11 deletion syndrome as aprototype, is that the microdeletions either uncover one or morespecific “risk” alleles, or that haploinsufficiency per se confersincreased risk.

Example 8 Exemplary Markers within 1 Linkage Disequilibrium Unit (1 LDU)

On-line public resources (HapMap.org) were used to identify exemplarySNPs that are in linkage disequilibrium with some of the SNPs describedherein, as follows:

rs738596

SNPs within 1 LDU of marker rs738596 in African American populationsinclude: rs5770635, rs17000207; in European American Populationsinclude: rs4823940, rs13053183, rs4824067, rs9628096; in Chinesepopulations include: rs5770579, rs8136613, rs4824067, rs17824774,rs5770632, rs9628096, rs5770634, rs5770635, rs9628100; and in Japanesepopulations include: rs2024698, rs9616622, rs5770581, rs4824067.

rs2073224

SNPs within 1 LDU of marker rs2073224 in African American populationsinclude: rs9616222, rs5769820, rs5769821, rs17178537, rs4823974,rs2064542; in European American populations include: rs9616222,rs5769820, rs5769821, rs17178537, rs6009133, rs6009134, rs4823974,rs2064542.

rs738615

SNPs within 1 LDU of marker rs738615 in Chinese populations include:rs4823908, rs2073225, rs761666, rs2064542; and in Japanese populationsinclude: rs9616409, rs4823908, rs2073225, rs761666, rs2064542.

rs848768

SNPs within 1 LDU of marker rs848768 in African American populationsinclude: rs12165304, rs9627698, rs9616561, rs9627966, rs12169496,rs12628115, rs11090946, rs848751, rs848750; in European AmericanPopulations include: rs2319174, rs739049, rs7287432, rs9616561,rs848764, rs848750; in Chinese populations include: rs739049, rs848764,rs12628115, rs11090946, rs848751, rs848750; and in Japanese populationsinclude: rs8135938, rs848751, rs848752.

rs737734

SNPs within 1 LDU of marker rs737734 in European American populationsinclude: rs5769607, rs5770363, rs714007, rs713997, rs5770369, rs7285315,rs2873922, rs2097363, rs7510746; and in Chinese populations include:rs5769607, rs8140231, rs713919, rs2097363, rs4824032, rs2187751.

rs134474

SNPs within 1 LDU of marker rs134474 in African American populationsinclude: rs6520121, rs17001084, rs17001087, rs3810643; in EuropeanAmerican Populations include: rs9616663, rs2873932, rs470019, rs470018,rs470017, rs134459, rs134458, rs134456.

rs763126

SNPs within 1 LDU of marker rs763126 in African American populationsinclude: rs135786, rs135787, rs135788, rs135789, rs135791, rs763124,rs135800, rs135804, rs8140984, rs135821, rs135827, rs135832, rs2319345,rs135833, rs135846, rs6009767, rs5769691, rs2071894, rs5770562,rs2071893, rs135854, rs135855, rs17001439, rs5770567, rs2007024,rs17182154, rs17001168, rs2187891, rs9616687, rs17001172, rs739247,rs2071890; in European American populations include: rs9616685,rs5769691, rs2071894, rs135853, rs5770562, rs2071893, rs135854,rs135855, rs5770567, rs2007024, rs739247, rs135861, rs2071890,rs12628438, rs10854876, rs6009782, rs135875, rs135876, rs135877; inChinese populations include: rs763124, rs135845, rs135846, rs135853,rs135827, rs135854, rs135855; rs2319345, rs9616685, rs5769691,rs2071894, rs5770562, rs135854, rs135855, rs5770567; and in Japanesepopulations include: rs135819, rs135827, rs135831, rs470058, rs2319345,rs135846, rs1008320, rs5769691, rs2071894, rs135853, rs2071893,rs2071892, rs135854, rs135855, rs135856, rs10854874.

rs138844

SNPs within 1 LDU of marker rs138844 in African American populationsinclude: rs138841, rs139818, rs10483250; in European Americanpopulations include: rs139818, rs138840, rs138841; in Chinesepopulations include: rs6009860, rs10483250, rs138841; and in Japanesepopulations include: rs10483250, rs6009870, rs5770689, rs138816,rs138820, rs138821, rs138823, rs138827, rs6009874, rs138840, rs138841,rs138843.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of determining a human subject's risk of developingschizophrenia (SZ), the method comprising: obtaining a sample comprisingDNA from the subject; determining the identity of both alleles ofrs1573726 in the sample; comparing the identity of the rs1573726 allelesin the subject to the alleles of rs1573726 in: (i) a relative of thesubject who is affected with SZ, and (ii) a relative of the subject whois not affected with SZ, wherein the presence of an allele at rs1573726in the subject that is the same as an allele in the relative who isaffected with SZ, and is not the same as the allele in the relative whois not affected with SZ, indicates that the subject has an increasedrisk of developing SZ.
 2. The method of claim 1, further comprisingdetermining the identity of both alleles of one or more of rs135221,rs763126, rs848768, rs2269523, rs738615, rs738596, rs738598, rs138844,or rs135819 in the subject, thereby determining a subject haplotype, andcomparing the subject haplotype to (i) a haplotype in a relative of thesubject who is affected with SZ, and (ii) a haplotype in a relative ofthe subject who is not affected with SZ, wherein the presence of ahaplotype in the subject that is the same as the haplotype in therelative who is affected with SZ, and is not the same as the haplotypein the relative who is not affected with SZ, indicates that the subjecthas an increased risk of developing SZ.
 3. The method of claim 1,further comprising: obtaining a sample comprising DNA from a relative ofthe subject who is affected with SZ, and determining the genotype ofrs1573726 in the sample; and obtaining a sample comprising DNA from arelative of the subject who is not affected with SZ, and determining theidentity of both alleles of rs1573726 in the sample.
 4. The method ofclaim 1, wherein the relative is a parent or sibling.
 5. The method ofclaim 1, wherein determining the identity of both alleles comprises:obtaining a sample comprising DNA from the subject; and determining theidentity of both alleles at rs1573726 in the sample.
 6. The method ofclaim 1, wherein the sample is obtained from the subject by a healthcare provider.
 7. The method of claim 1, wherein the sample is providedby the subject without the assistance of a health care provider.
 8. Themethod of claim 1, further comprising determining the presence orabsence of one or more additional markers associated with schizophrenia.9. The method of claim 1, wherein the subject is a patient having, or atrisk of, schizophrenia.
 10. The method of claim 1, wherein the subjectis suffering from early, intermediate or aggressive schizophrenia. 11.The method of claim 1, wherein the subject has one or more risk factorsassociated with SZ.
 12. The method of claim 11, wherein the risk factorsassociated with SZ include one or more of: a relative afflicted withschizophrenia, a genetically based phenotypic trait associated with riskfor SZ; deficits in working memory; and mixed-handedness, particularlyin females.
 13. The method of claim 12, wherein the subject has one ormore of a grandparent, parent, uncle or aunt, sibling, or child who hasor had SZ.
 14. The method of claim 12, wherein the genetically basedphenotypic is eye tracking dysfunction.
 15. The method of claim 1,wherein the subject is a child, fetus, or embryo, and one of therelatives of the subject has SZ.
 16. The method of claim 1, furthercomprising administering a treatment to a subject identified as being atincreased risk for developing SZ.
 17. The method of claim 16, whereinthe treatment is a pharmacological or psychosocial treatment for SZ. 18.The method of claim 1, further comprising stratifying a subjectpopulation for a clinical trial based on the genotype of rs1573726. 19.A method of selecting a human subject for inclusion or exclusion in aclinical trial, the method comprising: obtaining a sample comprising DNAfrom the subject; determining the identity of both alleles of rs1573726in the sample; comparing the identity of the alleles at rs1573726 in thesubject to the alleles of rs1573726 in: (i) a relative of the subjectwho is affected with SZ, and (ii) a relative of the subject who is notaffected with SZ, wherein the presence of an rs1573726 allele in thesubject that is the same as the allele in the relative who is affectedwith SZ, and is not the same as an allele in the relative who is notaffected with SZ, indicates that the subject has an increased risk ofdeveloping SZ; and including or excluding the subject if the genotypeindicates that the subject has an increased risk of developing SZ. 20.The method of claim 19, further comprising determining the identity ofboth alleles of one or more of rs135221, rs763126, rs848768, rs2269523,rs738615, rs738596, rs738598, rs138844, or rs135819, thereby providing asubject haplotype, and comparing the subject haplotype to: (i) ahaplotype in a relative of the subject who is affected with SZ, and (ii)a haplotype in a relative of the subject who is not affected with SZ,wherein the presence of a haplotype in the subject that is the same asthe haplotype in the relative who is affected with SZ, and is not thesame as the haplotype in the relative who is not affected with SZ,indicates that the subject has an increased risk of developing SZ; andincluding or excluding the subject if the haplotype indicates that thesubject has an increased risk of developing SZ.
 21. The method of claim19, wherein the clinical trial is of a treatment for SZ.